Plant extract compositions for forming protective coatings

ABSTRACT

Described herein are methods of preparing cutin-derived monomers, oligomers, or combinations thereof from cutin-containing plant matter. The methods can include heating the cutin-derived plant matter in a solvent at elevated temperature and pressure. In some preferred embodiments, the methods can be carried out without the use of additional acidic or basic species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2016/065917, filed Dec. 9,2016, which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 62/265,726, filed Dec. 10, 2015, the contents ofeach of which are incorporated by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to plant extract compositions and methodsof isolating plant-derived monomers, oligomers, and mixtures thereof forapplications in agricultural coating formulations.

BACKGROUND

Common agricultural products are susceptible to degradation anddecomposition (i.e., spoilage) when exposed to the environment. Suchagricultural products can include, for example, eggs, fruits,vegetables, produce, seeds, nuts, flowers, and/or whole plants(including their processed and semi-processed forms). Non-agriculturalproducts (e.g., vitamins, candy, etc.) are also vulnerable todegradation when exposed to the ambient environment. The degradation ofthe agricultural products can occur via abiotic means as a result ofevaporative moisture loss from an external surface of the agriculturalproducts to the atmosphere and/or oxidation by oxygen that diffuses intothe agricultural products from the environment and/or mechanical damageto the surface and/or light-induced degradation (i.e.,photodegradation). Furthermore, biotic stressors such as, for example,bacteria, fungi, viruses, and/or pests can also infest and decompose theagricultural products.

Conventional approaches to preventing degradation, maintaining quality,and increasing the life of agricultural products include refrigerationand/or special packaging. Refrigeration requires capital-intensiveequipment, demands constant energy expenditure, can cause damage orquality loss to the product if not carefully controlled, must beactively managed, and its benefits are lost upon interruption of atemperature-controlled supply chain. Special packaging can also requireexpensive equipment, consume packaging material, increase transportationcosts, and require active management. Despite the benefits that can beafforded by refrigeration and special packaging, the handling andtransportation of the agricultural products can cause surface abrasionor bruising that is aesthetically displeasing to the consumer and servesas points of ingress for bacteria and fungi. Moreover, the expensesassociated with such approaches can add to the cost of the agriculturalproduct.

The cells that form the aerial surface of most plants (such as higherplants) include an outer envelope or cuticle, which provides varyingdegrees of protection against water loss, oxidation, mechanical damage,photodegradation, and/or biotic stressors, depending upon the plantspecies and the plant organ (e.g., fruit, seeds, bark, flowers, leaves,stems, etc.). Cutin, which is a biopolyester derived from cellularlipids, forms the major structural component of the cuticle and servesto provide protection to the plant against environmental stressors (bothabiotic and biotic). The thickness, density, as well as the compositionof the cutin (i.e., the different types of monomers that form the cutinand their relative proportions) can vary by plant species, by plantorgan within the same or different plant species, and by stage of plantmaturity. The cutin-containing portion of the plant can also containadditional compounds (e.g., epicuticular waxes, phenolics, antioxidants,colored compounds, proteins, polysaccharides, etc.). This variation inthe cutin composition as well as the thickness and density of the cutinlayer between plant species and/or plant organs and/or a given plant atdifferent stages of maturation can lead to varying degrees of resistancebetween plant species or plant organs to attack by environmentalstressors (i.e., water loss, oxidation, mechanical injury, and light)and/or biotic stressors (e.g., fungi, bacteria, viruses, insects, etc.).

SUMMARY

Described herein are methods of preparing cutin-derived monomers,oligomers, or combinations thereof from cutin-containing plant matter.The method can comprise heating the cutin-containing plant matter in asolvent at elevated temperature and pressure.

Accordingly, in one aspect, the present disclosure provides a method ofpreparing cutin-derived monomers, oligomers, or combinations thereoffrom cutin-containing plant matter, comprising:

obtaining cutin from the cutin-containing plant matter;

adding the cutin to a solvent to form a first mixture, the solventhaving a boiling point at a first temperature at a pressure of oneatmosphere; and

heating the first mixture to a second temperature and second pressure,the second temperature being higher than the first temperature and thesecond pressure being higher than one atmosphere, to form a secondmixture comprising the cutin-derived monomers, oligomers, orcombinations thereof.

In another aspect, the present disclosure provides a method of forming aplant extract composition, comprising:

obtaining cutin from cutin-containing plant matter;

adding the cutin to a first solvent to form a first mixture, the firstsolvent having a first boiling point at a first temperature at apressure of one atmosphere;

heating the first mixture to a second temperature and second pressure,the second temperature being higher than the first temperature and thesecond pressure being higher than one atmosphere, to form a secondmixture comprising cutin-derived monomers, oligomers, or combinationsthereof;

separating the first solvent from the cutin-derived monomers, oligomers,or combinations thereof in the second mixture; and

dissolving the cutin-derived monomers, oligomers, or combinationsthereof in a second solvent.

In another aspect, the present disclosure provides a method of forming aplant extract composition, comprising:

obtaining cutin from cutin-containing plant matter;

adding the cutin to a first solvent to form a first mixture, the firstsolvent having a boiling point at a first temperature and firstpressure; and

heating the first mixture to a second temperature, the secondtemperature being higher than the first temperature, to form a secondmixture comprising cutin-derived monomers, oligomers, or combinationsthereof, wherein at least a portion of the cutin-derived monomers oroligomers in the second mixture are unsaturated.

In another aspect, the present disclosure provides a method of preparingcutin-derived monomers, oligomers, or combinations thereof fromcutin-containing plant matter, comprising:

obtaining cutin from the cutin-containing plant matter;

adding the cutin to a solvent to form a first mixture, the solventhaving a boiling point at a first temperature at a pressure of oneatmosphere;

heating the first mixture to a second temperature and second pressure,the second temperature being higher than the first temperature and thesecond pressure being higher than one atmosphere, to form a secondmixture comprising a first group of compounds of the Formula I:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently —H,—OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆alkynyl, —C₃-C₇ cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl,wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, or halogen;

R¹³ and R¹⁴ are each independently —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, or—C₁-C₆ alkynyl;

R¹¹ is —H, -glyceryl, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl,—C₃-C₇ cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, whereineach alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, or halogen;

R¹² is —OH, —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, wherein eachalkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —COOH, or —COOR¹¹; and

m, n, and o are each independently an integer in the range of 0 to 30,and 0≦m+n+o≦30.

In another aspect, the present disclosure provides a method of preparinga composition comprising esters of cutin-derived fatty acids. The methodincludes obtaining cutin from cutin-containing plant matter, and addingthe cutin to a solvent to form a mixture, the solvent having a boilingpoint at a first temperature at a pressure of one atmosphere. The methodfurther includes heating the mixture to a second temperature and secondpressure, the second temperature being higher than the first temperatureand the second pressure being higher than one atmosphere, therebyforming the composition comprising the esters.

In another aspect, the present disclosure provides a method of preparinga composition comprising cutin-derived free fatty acid monomers,oligomers, or combinations thereof. The method includes obtaining cutinfrom cutin-containing plant matter, adding the cutin to water to form amixture. The method further includes heating the mixture from a firsttemperature and first pressure to a second temperature and secondpressure, the second temperature being higher than the boiling point ofwater at one atmosphere and the second pressure being higher than oneatmosphere, thereby forming the composition comprising the cutin-derivedfree fatty acid monomers, oligomers, or combinations thereof.

In another aspect, the present disclosure provides a method of forming aprotective coating. The method includes extracting a composition from acuticle layer of a first plant species, the composition including aplurality of cutin-derived monomers, oligomers, or combinations thereof,and disposing the composition on a second plant species which is eitherthe same or different from the first plant species to form theprotective coating over the second plant species.

In another aspect, the present disclosure provides a method of forming aprotective coating. The method includes extracting a composition from acuticle layer of plant matter of a first plant, the compositionincluding a plurality of cutin-derived free fatty acid monomers oroligomers, fatty acid esters, or combinations thereof, and disposing thecomposition on plant matter of a second plant different from the firstplant, thereby forming the protective coating over the plant matter ofthe second plant.

In another aspect, the present disclosure provides a method of preparingcutin-derived monomers, oligomers, or combinations thereof fromcutin-containing plant matter, comprising:

obtaining cutin from the cutin-containing plant matter;

adding the cutin to a solvent to form a first mixture, the solventhaving a boiling point at a first temperature at a pressure of oneatmosphere;

heating the first mixture to a second temperature and second pressure,the second temperature being higher than the first temperature and thesecond pressure being higher than one atmosphere, to form a secondmixture comprising a first group of compounds of the Formula II:

wherein:

R¹, R², R⁴ and R⁵ are each independently —H, —OR¹¹, —NR¹¹R¹², —SR¹¹,halogen, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, wherein eachalkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with —OR¹¹, —NR¹¹R¹², —SR¹¹, or halogen;

R¹¹ and R¹² are each independently —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, or—C₁-C₆ alkynyl;

the symbol

represents an optionally single or cis or trans double bond;

R³ is —OH and R^(3′) is selected from the group consisting of —H, —C₁-C₆alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and aryl when

between R³ and R^(3′) is a single bond, and R³ and R^(3′) are absentwhen

between R³ and R^(3′) represents a double bond;

R⁶ is —OH and R^(6′) is selected from the group consisting of —H, —C₁-C₆alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and aryl when

between R⁶ and R^(6′) is a single bond, and R⁶ and R^(6′) are absentwhen

between R⁶ and R^(6′) represents a double bond;

n is an integer in the range of 0 to 11;

m is an integer in the range of 0 to 25; and

0≦m+n≦25.

In another aspect, the present disclosure provides a method of preparingcutin-derived monomers, oligomers, or combinations thereof fromcutin-containing plant matter, comprising:

obtaining cutin from the cutin-containing plant matter;

adding the cutin to a solvent to form a first mixture, the solventhaving a boiling point at a first temperature at a pressure of oneatmosphere;

heating the first mixture to a second temperature and second pressure,the second temperature being higher than the first temperature and thesecond pressure being higher than one atmosphere, to form a secondmixture comprising a first group of compounds of the Formula III:

wherein:

R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each independently, ateach occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl,wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, orhalogen;

R³, R⁴, R⁷, and R⁸ are each independently, at each occurrence, —H,—OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl whereineach alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, or halogen; or

R³ and R⁴ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle; and/or

R⁷ and R⁸ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle;

R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl;

the symbol

represents a single bond or a cis or trans double bond;

n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

m is 0, 1, 2 or 3;

q is 0, 1, 2, 3, 4 or 5; and

r is 0, 1, 2, 3, 4, 5, 6, 7 or 8; and

R is selected from —H, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl,—C₃-C₇ cycloalkyl, aryl, 1-glycerol, 2-glycerol, or heteroaryl.

Methods and formulations described herein can each include one or moreof the following steps or features, either alone or in combination withone another. The second mixture can be cooled to a third temperature,the third temperature being lower than the second temperature. The stepof cooling the second mixture can further comprise reducing the secondpressure to a third pressure, the third pressure being below the secondpressure. The third temperature can be lower than the first temperature.The third pressure can be substantially the same as the first pressure.The third pressure can be about one atmosphere. The method can furthercomprise separating the solvent from the second mixture to isolate thecutin-derived monomers, oligomers, or combinations thereof. The secondtemperature can be at least 5% higher than the first temperature. Thesecond pressure can be sufficiently high to maintain at least a portionof the solvent in a liquid phase at the second temperature. The secondpressure can be higher than the first pressure. The cutin can be atleast partially separated from a non-cutin containing portion of theplant matter prior to adding the cutin to the solvent.

The process of forming the second mixture can further result in theproduction of unsaturated fatty acids. The process of forming the secondmixture can result in the production of both saturated and unsaturatedfatty acids. The production of unsaturated fatty acids can be the resultof elimination of a hydroxy group bound to the fatty acid chain. Aconcentration of saturated fatty acids can be substantially higher thana concentration of unsaturated fatty acids in the second mixture. Theprocess of forming the second mixture can further result in theproduction of unsaturated fatty acids. The process of forming the secondmixture can result in the production of both saturated and unsaturatedfatty acid esters. The production of unsaturated fatty acid esters canbe the result of elimination of a hydroxy group bound to the fatty acidchain. A concentration of saturated fatty acid esters can besubstantially higher than a concentration of unsaturated fatty acidesters in the second mixture.

The heating of the first mixture can be performed while the firstmixture is in a vessel, and the method can further comprise injecting agas or liquid into the vessel during the heating of the first mixture. Adepolymerization reaction at the second temperature can result inuncharged cutin-derived monomers, oligomers, or combinations thereof.The solvent can be selected such that concentrations of reactive anionsand reactive cations dispersed therein while the solvent is held at thesecond temperature are sufficient to at least partially depolymerize thecutin and to result in uncharged cutin-derived monomers, oligomers, orcombinations thereof. A rate of depolymerization of the cutin in thesolvent can be at least 100 times greater at the second temperature andthe second pressure than at the first temperature at a pressure of oneatmosphere. The cutin-derived monomers, oligomers, or combinationsthereof in the second mixture can be substantially soluble in thesolvent at about 298 K. The second temperature can be greater than about393 K. The second temperature can be at least about 498 K. The solventcan be selected from the group consisting of water, glycerol, methanol,ethanol, liquid CO₂, and supercritical CO₂, or a combination thereof.The solvent can be a nucleophilic solvent. The solvent can be water. Thefirst mixture can further include a co-solvent. The co-solvent cancomprise CO₂. The solvent can be substantially free of added acid orbase.

The second temperature can be at least about 498K and the secondpressure can be at least about 360 psi, and the first mixture can beheld at the second temperature and second pressure for at least abouteight hours. The second temperature can be at least about 523K and thesecond pressure can be at least about 575 psi, and the first mixture canbe held at the second temperature and second pressure for at least abouttwo hours. The first mixture can further include a reactive additive.The reactive additive can be an enzyme, NaOH, Na₂CO₃, acetic acid, a pHmodifier, or a combination thereof.

The cutin can be at least partially separated from a non-cutincontaining portion of the plant matter prior to adding the cutin to thefirst solvent. The cutin-derived monomers, oligomers, or combinationsthereof in the second mixture can be filtered from non-depolymerizedresidue and separated from the first solvent. At least a portion of thecutin-derived monomers, oligomers or combinations thereof can beunsaturated fatty acids. The method can further comprise hydrogenatingthe unsaturated fatty acids before adding them to the second solvent.The unsaturated fatty acids can be dissolved in the second solventwithout hydrogenation. At least a portion of the cutin-derived monomers,oligomers or combinations thereof can be unsaturated fatty acid esters.The method can further comprise hydrogenating the unsaturated fatty acidesters before adding them to the second solvent. The unsaturated fattyacid esters can be dissolved in the second solvent withouthydrogenation.

The method can further comprise separating the first solvent from thecutin-derived monomers, oligomers, or combinations thereof in the secondmixture, and hydrogenating the unsaturated cutin-derived monomers andoligomers to form a third mixture comprising cutin-derived monomers,oligomers, or combinations thereof, wherein substantially all of thecutin-derived monomers or oligomers in the third mixture are saturated.The unsaturated cutin-derived monomers and oligomers in the secondmixture can be separated from saturated cutin-derived monomers andoligomers in the second mixture prior to hydrogenation. In someembodiments, the unsaturated cutin-derived monomers and oligomers in thesecond mixture are not separated from saturated cutin-derived monomersand oligomers in the second mixture prior to hydrogenation. Betweenabout 10% and 98% of the cutin-derived monomers and oligomers in thesecond mixture can be unsaturated. Between 0.5% and 30%, greater than 0%but less than 40%, less than 10%, between about 1% and 98%, between 1%and 99%, or substantially all of the cutin-derived monomers andoligomers in the second mixture can be unsaturated. The method canfurther comprise dissolving the third mixture in a second solvent.

The method can further produce compounds of the Formula II:

wherein:

R¹, R², R⁴ and R⁵ are each independently —H, —OR¹¹, —NR¹¹R¹², —SR¹¹,halogen, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, wherein eachalkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with —OR¹¹, —NR¹¹R¹², —SR¹¹, or halogen;

R¹¹ and R¹² are each independently —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, or—C₁-C₆ alkynyl;

the symbol

represents an optionally single or cis or trans double bond;

R³ is —OH and R^(3′) is selected from the group consisting of —H, —C₁-C₆alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and aryl when

between R³ and R^(3′) is a single bond, and R³ and R^(3′) are absentwhen

between R³ and R^(3′) represents a double bond;

R⁶ is —OH and R^(6′) is selected from the group consisting of —H, —C₁-C₆alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and aryl when

between R⁶ and R^(6′) is a single bond, and R⁶ and R^(6′) are absentwhen

between R⁶ and R^(6′) represents a double bond;

n is an integer in the range of 0 to 11;

m is an integer in the range of 0 to 25; and

0≦m+n≦25.

The method can further produce a second group of compounds of theFormula I, the second group of compounds of Formula I being differentfrom the first group of compounds of the Formula I. The method canproduce compounds of Formula II that can then be transformed intocompounds of Formula I, for example through conventional synthetictransformation, wherein the compounds of Formula I that are therebyformed may differ from compounds of Formula I produced directly bythermal depolymerization. For instance, an acid formed by a hydrothermaldepolymerization method as set forth herein can be subsequentlyconverted to an ester. The forming of the protective coating cancomprise causing at least some of the cutin-derived monomers, oligomers,or combinations thereof to cross-link on the second plant species. Themethod can further include chemically modifying the cutin-derivedmonomers, oligomers, or combinations thereof prior to disposing thecomposition on the second plant species. Chemically modifying thecutin-derived monomers, oligomers, or combinations thereof can compriseglycerating the cutin-derived monomers, oligomers, or combinationsthereof (e.g., forming a glycerol ester of the corresponding fatty acidor ester). A chemical composition of the protective coating can bedifferent from a chemical composition of a cuticle layer of the secondplant species. The extracting of the composition from the cuticle layerof the first plant species can comprise obtaining cutin from the cuticlelayer of the first plant species, adding the cutin to a solvent to forma first mixture, the solvent having a boiling point at a firsttemperature at a pressure of one atmosphere, and heating the firstmixture to a second temperature and second pressure, the secondtemperature being higher than the first temperature and the secondpressure being higher than one atmosphere, to form a second mixturecomprising the cutin-derived monomers, oligomers, or combinationsthereof. The method can further comprise modifying the compounds ofFormula II to form compounds of Formula I, where Formula II and FormulaI are as previously described. The method can further comprise modifyingthe compounds of Formula III to form compounds of Formula I, whereFormula III and Formula I are as previously described.

The solvent in which the cutin is added can comprise ethanol, and theesters of the resulting composition can comprise ethyl esters. Thesolvent in which the cutin is added can comprise methanol, and theesters of the resulting composition can comprise methyl esters. Thesolvent in which the cutin is added can comprise glycerol, and theesters of the resulting composition can comprise glyceryl esters.

As used herein, the term “substrate” refers to any object or materialover which a coating is formed or material is deposited. In particularimplementations, the substrate is edible to humans, and the coating isan edible coating. Examples of edible substrates include agriculturalproducts and foods such as fruits, vegetables, produce, seeds, nuts,beef, poultry, and seafood. Although the coatings can be formed over theentire outer surface of the substrate, in some implementations thecoatings can cover a portion of the outer surface of the substrate. Thecoatings can also include apertures or porous regions which expose aportion of the outer surface of the substrate.

As used herein, “plant matter” refers to any portion of a plant, forexample, fruits (in the botanical sense, including fruit peels and juicesacs), leaves, stems, barks, seeds, flowers, or any other portion of theplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a phase diagram of H₂O.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the chemical structure of10,16-dihydroxyhexadecanoic acid, 10,18-dihydroxyoctadecanoic acid,9,16-dihydroxyhexadecanoic acid, 9,18-dihydroxyoctadecanoic acid,9,10,16-trihydroxyhexadecanoic acid, 9,10,18-trihydroxyoctadecanoicacid, respectively.

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I are byproducts that canresult from the decomposition of 10,16-dihydroxyhexadecanoic acidmonomers and/or oligomers during a thermal depolymerization process.

FIGS. 4A and 4B are qualitative plots of the relative concentrations ofdirect depolymerization products and unsaturated indirect byproducts asa function of time that result from thermal depolymerization of cutinwhen water is used as the solvent.

FIGS. 5A, 5B and 5C are chemical compositions of molecules that can beformed from unsaturated indirect byproducts of thermal depolymerizationof cutin.

FIGS. 6A and 6B are esters that can be formed by thermaldepolymerization of cutin in a solvent that includes ethanol.

FIGS. 6C and 6D are esters that can be formed by thermaldepolymerization of cutin in a solvent that includes methanol.

FIGS. 6E, 6F, 6G, and 6H are esters that can be formed by thermaldepolymerization of cutin in a solvent that includes glycerol.

FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, and 7I are esters that can beformed by thermal depolymerization of cutin in a solvent that includesethanol.

FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I are esters that can beformed by thermal depolymerization of cutin in a solvent that includesmethanol.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, and 9I are esters that can beformed by thermal depolymerization of cutin in a solvent that includesglycerol.

FIGS. 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, and 10I are esters thatcan be formed by thermal depolymerization of cutin in a solvent thatincludes glycerol.

FIG. 11 illustrates a process for forming a protective coating.

FIG. 12A illustrates a plot of the amount of crude cutin monomers and/oroligomers recovered by thermal depolymerization of cutin in water forvarious temperatures and reaction durations.

FIG. 12B illustrates a plot of the amount of product (saturated productand/or unsaturated byproduct) isolated from the crude cutin monomersand/or oligomers in the heptane precipitate after soxhlet extraction forvarious temperatures and residence times.

FIG. 12C is a plot of the amount of unsaturated byproduct isolated fromthe crude cutin monomers and/or oligomers in the heptane supernatantafter soxhlet extraction for various temperatures and residence times.

FIG. 12D illustrates a plot of the amount of saturated product isolatedfrom the crude cutin monomers and/or oligomers in the ethyl acetatesupernatant after soxhlet extraction for various temperatures andresidence times.

FIG. 13 is a table indicating the nature of products isolated aftersoxhlet extraction using heptane and ethyl acetate after thermalhydrolysis in water at various temperatures and reaction durations.

FIG. 14A is a plot of percentage of crude product recovered by thermaldepolymerization of cutin in ethanol for various temperatures andresidence times.

FIG. 14B is a plot of percentage of product recovered from a heptaneprecipitate during thermal depolymerization of cutin in ethanol forvarious temperatures and residence times.

FIG. 14C is a plot of percentage of product recovered from a heptanesupernatant during thermal depolymerization of cutin in ethanol forvarious temperatures and residence times.

FIG. 15 is a table indicating which products were recovered from theheptane precipitate and from the heptane supernatant at variousdepolymerization temperatures and residence times for thermaldepolymerization of cutin in ethanol.

FIG. 16 is a plot of mass loss rates of avocados coated withcompositions formed by methods described herein.

FIG. 17 shows UPLC traces of crude product recovered after hydrothermaldepolymerization at 548 K for one hour.

FIG. 18 shows UPLC traces and mass spectrometry analysis of10,16-dihydroxyhexadecanoic acid recovered after hydrothermaldepolymerization at 548 K for one hour before extraction.

FIG. 19 shows UPLC traces and mass spectrometry analysis of unsaturatedfatty acid recovered after hydrothermal depolymerization at 548 K forone hour before extraction.

FIG. 20 shows UPLC traces and mass spectrometry analysis of10,16-dihydroxyhexadecanoic acid recovered from the heptane precipitateafter hydrothermal depolymerization at 548 K for one hour and soxhletextraction with heptane.

FIG. 21 shows UPLC traces and mass spectrometry analysis of unsaturatedfatty acid recovered from heptane supernatant after hydrothermaldepolymerization at 548 K for one hour and soxhlet extraction withheptane.

FIG. 22 shows UPLC traces and mass spectrometry analysis of10,16-dihydroxyhexadecanoic acid recovered from the ethyl acetatesupernatant after hydrothermal depolymerization at 548 K for one hourand soxhlet extraction with ethyl acetate.

FIG. 23 shows UPLC traces of crude product recovered after ethanolysisdepolymerization at 548 K for four hours.

FIG. 24 shows UPLC traces and mass spectrometry analysis of ethyl10,16-dihydroxyhexadecanoate recovered from the heptane supernatantafter ethanolysis depolymerization at 548 K for four hours and soxhletextraction with heptane.

FIG. 25 shows UPLC traces and mass spectrometry analysis of unsaturatedfatty acid ethyl ester recovered from the heptane supernatant afterethanolysis depolymerization at 548 K for four hours and soxhletextraction with heptane.

Like reference symbols in the various figures indicate like elements.

DETAILED DESCRIPTION

The biopolyester cutin forms the main structural component of thecuticle that composes the aerial surface of most land plants and plays asignificant role in providing plants a protective barrier against bothabiotic and biotic stressors. The thickness, density, as well as thecomposition of the cutin (i.e., the different types of monomers thatform the cutin and their relative proportions) can vary by plantspecies, by plant organ within the same or different plant species, andby stage of plant maturity. These variations can define the amount,degree, or quality of protection (and degree of plasticity) offered bythe cutin layer to the plant or plant organ against environmental and/orbiotic stressors. Cutin is formed from a mixture of polymerized mono-and/or polyhydroxy fatty acids and embedded cuticular waxes. The hydroxyfatty acids of the cuticle layer form tightly bound networks with highcrosslink density, thereby acting as a barrier to moisture loss andoxidation, as well as providing protection against other environmentalstressors.

Described herein are methods of preparing and forming plant extractcompositions from plant matter. The plant extract compositions areformed from decomposition (e.g., depolymerization) of cutin or othercrosslinked polymers (e.g., polyesters), and include hydroxy fatty acidsand hydroxy fatty esters (as well as their oligomers and mixturesthereof) found in the cuticle layer or other crosslinked polymernetwork. The plant extract compositions can subsequently be applied toother plant or agricultural products in order to form a protectivecoating over the products, or to enhance or modify existing coatings(either naturally occurring or deposited coatings) which are on theouter surface of the products. The applied coatings can, for example,serve to protect the products from biotic stressors such as bacteria,fungi, viruses, and/or pests. The applied coatings can also (oralternatively) serve to increase the shelf life of produce withoutrefrigeration, and/or to control the rate of ripening of produce. Themethods of forming the plant extract compositions can result in thecompositions being substantially free of other accompanyingplant-derived compounds (e.g., proteins, polysaccharides, phenols,lignans, aromatic acids, terpenoids, flavonoids, carotenoids, alkaloids,alcohols, alkanes, and aldehydes), thereby improving the efficacy of thesubsequently formed protective coatings.

As described above, the monomer and/or oligomer units of the plantextract compositions can be obtained from cutin found in plant matter.Plant matter typically includes some portions that contain cutin and/orhave a high density of cutin (e.g., fruit peels, leaves, shoots, etc.),as well as other portions that do not contain cutin or have a lowdensity of cutin (e.g., fruit flesh, seeds, etc.). The cutin-containingportions can be used to produce plant extract compositions comprisingcutin-derived monomers and/or oligomers, and can also include otherconstituents such as proteins, polysaccharides, phenols, lignans,aromatic acids, terpenoids, flavonoids, carotenoids, alkaloids,alcohols, alkanes, and aldehydes. The low cutin density ornon-cutin-containing portions can often lack the monomer and/or oligomerunits, or otherwise include a much lower ratio of monomer and/oroligomer units to the other constituents as compared to the higherdensity cutin-containing portions.

Methods described herein for forming plant extract compositions caninclude first separating (or at least partially separating)cutin-containing portions of plant matter from non-cutin-containingportions, and obtaining cutin from the cutin-containing portions (e.g.,when the cutin-containing portion is a fruit peel, the peel is separatefrom the fruit body, and/or the cutin is separated from the peel). Thecutin, or peel containing cutin, is then depolymerized (or at leastpartially depolymerized) using a thermal process, described in detailbelow, in order to obtain a mixture including a plurality of fatty acidor esterified cutin-derived monomers, oligomers, or combinationsthereof. The thermal process for depolymerization causes most of orsubstantially all of (e.g., at least 95% of) the resulting monomersand/or oligomers of the mixture to be protonated or rendered neutral(i.e., uncharged) without requiring any additional processes (e.g.,acidification). In other words, the depolymerization processes describedherein can be carried out in the absence of added base or acid. Thisresults in the monomers and/or oligomers being in a state such that theycan subsequently be chemically modified to provide compounds whoseproperties can be tailored for specific applications. For example,oxygen and water barrier properties of subsequently formed coatings canbe controlled by chemically modifying the monomers and/or oligomers, andsuch modifications may require that the monomers and/or oligomers firstbe protonated or rendered neutral. Furthermore, the chemicalmodification of the monomers and/or oligomers can be tailored to changethe solubility of the extract composition in order to allow for expandedoptions for coating deposition. Finally, the mixture including the freefatty acid and/or free fatty ester monomer and/or oligomer units isdissolved in another solvent to form a solution, thereby resulting in aplant extract composition suitable for coating applications (e.g.,agricultural coating applications). Optionally, prior to forming theplant extract composition, the free fatty acid and/or free fatty estermonomer and/or oligomer units of the mixture are activated or modified(e.g., glycerated), for example to form a mixture of1-monoacylglycerides and/or 2-monoacylglycerides, and the mixture ofmodified monomer and/or oligomer units (e.g., 1-monoacylglyceridesand/or 2-monoacylglycerides) is dissolved in a solvent to form asolution, thereby resulting in the plant extract composition.

As described above, to form a cutin-derived plant extract compositionsuitable for coating applications, cutin-containing portions of plantmatter are first separated (or at least partially separated) fromnon-cutin-containing portions. This can be achieved by a number ofmethods, either alone or in combination with one another. For example,the plant matter can be thermally and/or mechanically and/orenzymatically and/or chemically treated to at least partially separatethe cutin-containing portion from the non-cutin-containing portion. Or,the plant matter can be subjected to elevated temperature and/orpressure in an aqueous medium (e.g., as in pressure cooking) topartially separate the cutin-containing portion from thenon-cutin-containing portion of the plant matter. Alternatively, theplant matter may be subjected to lower temperatures (e.g., as infreezing) to partially separate the cutin-containing portion from thenon-cutin-containing portion of the plant matter. The plant matter mayalso be subjected to sonication in an aqueous medium to partiallyseparate the cutin-containing portion from the non-cutin-containingportion of the plant matter. Optionally, the cutin-containing portioncan be heated in a mixture of ammonium oxalate and oxalic acid to aidseparation of the cutin from the non-cutin-containing portion (i.e., theremainder of the cuticle and unwanted plant matter). Optionally, thisseparation can be achieved (or assisted) enzymatically using enzymescapable of hydrolyzing ester bonds and/or alternatively using enzymescapable of breaking down polysaccharides that comprise thenon-cutin-containing portion of the plant. The cutin-containing portioncan optionally be refluxed in at least one organic solvent (such aschloroform and/or methanol) to remove residual waxes and/or anyremaining soluble components from the cutin. Alternatively, removal ofresidual waxes and remaining soluble components can be achieved usingliquid or supercritical CO₂.

After separating (or at least partially separating) the cutin-containingportions of the plant matter from non-cutin-containing portions, thecutin (or cutin-containing component) obtained from the plant matter isthen added to a solvent to form a first mixture. The solvent can, forexample, be a nucleophilic solvent such as water, methanol, ethanol,glycerol, or combinations thereof. The first mixture may optionallyinclude a co-solvent such as water, methanol, ethanol, glycerol, liquidCO₂, or supercritical CO₂. The co-solvent can also be nucleophilic(e.g., when the co-solvent is water, methanol, glycerol, or ethanol), oralternatively the co-solvent may not be nucleophilic (e.g., when theco-solvent is liquid CO₂ or supercritical CO₂). Optionally, the solventcan serve as a reactive modifier. Optionally, the co-solvent can serveas a catalyst to depolymerize the cutin containing portion, and thesolvent can serve to transesterify the intermediate depolymerizedproduct. Optionally, the solvent can also include a reactive additive orprocessing aid such as an enzyme, NaOH, Na₂CO₃, acetic acid, another pHmodifier, or combinations thereof. If a reactive additive or processingaid is included, the concentration of the reactive additive orprocessing aid can be sufficiently low such that substantialdepolymerization of the cutin does not occur in the absence of thethermal conditions described below. Alternatively, the reactive additiveor processing aid can be included in sufficiently high concentration tocatalyze the depolymerization process, and the thermal process can beperformed to further enhance or increase the reaction rate of thedepolymerization process.

The first mixture including the cutin-containing component in thesolvent (and optionally the co-solvent and/or the reactive additive orprocessing aid) is then subjected to elevated temperature and pressure(i.e., a thermal process) for a sufficiently long time to allow thecutin (or cutin-containing component) to at least partially depolymerizeinto cutin-derived monomers, oligomers, esters thereof, or a combinationthereof, thereby forming a second mixture comprising the cutin-derivedmonomers, oligomers, esters thereof, or a combination thereof. Forexample, the first mixture can be placed in a vessel such thatapproximately 50-100% of the vessel volume is filled with the firstmixture, and the vessel and enclosed first mixture can then be sealedand heated to a temperature greater than the boiling point of thesolvent at atmospheric pressure (i.e., the temperature at which thesolvent would have boiled at if it had been maintained at 1 atm). Forexample, the vessel and enclosed first mixture can be heated above atemperature which is at least 5%, at least 10%, at least 15%, at least20% at least 25% at least 30%, at least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 100% greater than theboiling point of the solvent at atmospheric pressure (i.e., at 1 atm).In some implementations, the solvent is water, and the mixture is heatedto a temperature of at least 393 K, at least 423 K, at least 453 K, atleast 473 K, at least 498K, or at least 523 K. In some implementations,the mixture is maintained below a temperature of 1000 K.

Due to thermal expansion of the mixture (and any air or other gas orfluid in the sealed vessel), as well as vaporization of the solvent, thepressure within the vessel increases at the elevated temperature, andcan, for example, be greater than 1 atm, greater than 5 atm, greaterthan 10 atm, or greater than 100 atm. In some implementations, thepressure self-adjusts to a value at or near that of the liquid-gastransition point of the solvent at the elevated temperature, as furtherdescribed below. The pressure can optionally be further increased withinthe vessel, for example by pumping nitrogen gas into the vessel, or bypumping solvent into the vessel with pressure maintained by a backflowor pressure regulator. The first mixture can be held at the elevatedtemperature and pressure for a predetermined amount of time sufficientto cause depolymerization of the cutin-containing component in the firstmixture into cutin-derived monomers, oligomers, esters, or combinationsthereof, thereby forming the second mixture. After the first mixture isheld at the elevated temperature and pressure for the predeterminedamount of time to form the second mixture, the second mixture is cooledand the pressure released.

Abbreviations used herein include DHPA [10,16-dihydroxyhexadecanoic acidacid], MEHPA [(E/Z)-16-hydroxyhexadec-9-enoic acid], EtDHPA [ethyl10.16-dihydroxyhexadecanoate], and EtMEHPA [ethyl(E/Z)-16-hydroxyhexadec-9-enoate].

As used herein, the term “temperature” refers to absolute temperature,as measured in units of Kelvin (K). Accordingly, if a first temperatureis X % greater than a second temperature, the first temperature(measured in K) is at least (1+X/100) times the second temperature(measured in K). For example, in the case of water, which has a boilingpoint of 373.15 K at 1 atm, a temperature which is at least 5% greaterthan the boiling point of water at 1 atm corresponds to a temperaturegreater than 391.81 K (i.e., greater than 1.05×373.15 K).

In some implementations, the cutin-derived monomers, oligomers, estersor combinations thereof resulting from the thermal depolymerizationprocess are soluble in the solvent at the elevated temperature at whichthe thermal process is carried out, and also at the temperature at whichthe second mixture is subsequently cooled to (typically roomtemperature). This can be the case when the solvent is ethanol ormethanol, for example. In other implementations, the cutin-derivedmonomers, oligomers, esters, or combinations thereof resulting from thethermal depolymerization process are insoluble in the solvent at theelevated temperature at which the thermal process is carried out, andalso at the temperature at which the second mixture is subsequentlycooled to. In still other implementations, the cutin-derived monomers,oligomers, esters, or combinations thereof resulting from the thermaldepolymerization process are soluble in the solvent at the elevatedtemperature at which the thermal process is carried out, but areinsoluble at the temperature at which the second mixture is subsequentlycooled to (e.g., room temperature or about 300 K). In this case, themonomers, oligomers, esters, or combinations thereof which are dissolvedat the elevated temperature precipitate as the second mixture is cooledto room temperature, resulting in the monomers, oligomers, esters, orcombinations thereof being suspended in the solvent, and for examplebeing intermixed with other non-cutin plant matter as a solid char. Whenwater is used as the solvent, for example, the cutin-derived monomers,oligomers, esters, or combinations thereof typically may be insoluble atroom temperature, but may or may not be soluble at the elevatedtemperature and pressure, depending on the specific plant from which thecutin is sourced.

As previously described, while heating the first mixture to and holdingit at the elevated temperature, the pressure within the vessel can beincreased to ensure that at least 50% of the solvent is maintained inthe liquid phase. For example, consider the phase diagram 100 of water(H₂O) shown in FIG. 1. When water, which has a boiling point of about373 K at 1 atm, is used as the solvent in the first mixture, the firstmixture can be heated to a second temperature greater than 373 K (e.g.,temperature 104 in FIG. 1) and maintained substantially in the liquidphase if the pressure is also increased to a second pressure which isgreater than or about equal to that at the liquid-gas phase boundary forthe particular temperature. For example, when the first mixture isheated to temperature 104, the pressure can increase such that the waterin the mixture is at point 102 in the phase diagram 100 of FIG. 1.

As previously described, the increased pressure required to maintain atleast 50% of the solvent in the liquid phase at the elevated temperaturecan be achieved, for example, by sealing the vessel such that thepressure in the vessel increases as the temperature is increased due tothermal expansion of the mixture, vaporization of some portion of thesolvent, and/or thermal expansion of any air or other gas or fluid inthe sealed vessel. The resulting pressure in the vessel can self-adjustso that it is approximately at the liquid-gas transition point. Forexample, the pressure can adjust to a value that is within 1%, within 2%or within 5% of the liquid-gas transition point at the elevatedtemperature. The exact value of the resulting pressure depends at leastpartially on the percent volume of the vessel which is filled with themixture. However, if the fill ratio is too large, e.g., if the fillratio approaches 1, the pressure may become too high for the vessel tosupport mechanically. Accordingly, in some implementations, more than50% but less than 99% of the vessel volume is filled with the mixture.For example, between 60% and 95% or between 70% and 95% of the vesselvolume may be filled with the mixture. Furthermore, in someimplementations the pressure in the vessel at the elevated temperatureis sufficient to maintain at least 60%, at least 70%, at least 80%, atleast 90%, or substantially all of the solvent in the liquid phase.

Additionally, the pressure within the vessel can be further increased bypumping additional gas or liquid, for example nitrogen, into the vessel.This can allow the pressure within the vessel to be directly controlledby means of a pressure regulator or backflow preventer in order tobetter optimize the depolymerization process, which is further describedbelow. Furthermore, if the liquid contains additional material to beprocessed, a flow through reaction may be implemented.

The solvent in which the cutin-containing portion is depolymerized canbe selected such that the cutin-containing portion is not substantiallydepolymerized at room temperature (e.g., at 300 K) and/or is notsubstantially depolymerized or has a very low depolymerization rate attemperatures below the boiling point at 1 atm. Thus, in many cases, therate of depolymerization is only high enough for substantialdepolymerization to occur when the temperature is raised substantiallyabove the atmospheric boiling point of the solvent (e.g., at least 5% orat least 10% above the atmospheric boiling point). As such, in order tomaintain the solvent in the liquid phase so that depolymerization canoccur, the pressure in the vessel is raised accordingly, as previouslydescribed.

Substantial depolymerization of the cutin-containing portion canalternatively be achieved at room temperature or at temperatures belowthe atmospheric boiling point of the solvent by acidifying or alkalizingthe solvent, for example by adding metal hydroxides to the solvent.However, the thermal depolymerization process described above canprovide certain advantages over such a process. For example, the thermaldepolymerization process can self generate both reactive anions andcations in the solvent, thereby depolymerizing the cutin intomonomer/oligomer units. The depolymerization products are inherentlyuncharged, which may be necessary or desirable for subsequentlymodifying (e.g., esterifying or glycerating) the monomer/oligomer unitsand/or for forming protective coatings from the monomer/oligomer units.Conversely, depolymerization in a strong base typically does not resultin the monomer and/or oligomer products of the depolymerization processbeing uncharged. As such, additional steps in which the monomer/oligomerproducts are neutralized may be required in order to form protectivecoatings having desirable properties from the monomer/oligomer units.Additionally, omitting the use of strong acids and/or bases from thedepolymerization process can result in the process being recognized asfully organic.

The specific temperature(s) at which the thermal depolymerizationprocess is carried out, as well as the composition of the solvent, canbe selected such that concentrations of reactive anions and reactivecations in the solvent while the solvent is held at the elevatedtemperature (and corresponding elevated pressure) are sufficient todepolymerize the cutin. The temperature may further be selected suchthat the rate of depolymerization of the cutin in the solvent is atleast 100 times greater at the elevated temperature (and correspondingelevated pressure) than at standard temperature and pressure. Forexample, when H₂O is used as the solvent for the thermaldepolymerization process, the temperature can be greater than 393 K, forexample at least 413 K, at least 433 K, at least 453 K, at least 473K,greater than 483 K, greater than 498 K, greater than 513 K, greater than523 K, between 473 K and 523 K, or between 493 K and 533 K. In someimplementations, the thermal depolymerization process is carried outusing supercritical H₂O as the solvent and optionally usingsupercritical CO₂ as a co-solvent, in which case the elevatedtemperature is greater than 647 K and the pressure is greater than 218atm (e.g., between 218 and 1000 atm). In some implementations, thethermal depolymerization process is carried out using supercriticalethanol as the solvent and optionally using supercritical CO₂ as aco-solvent, in which case the elevated temperature can be greater than514 K and the pressure can be greater than 60.6 atm (e.g., between 60.6and 1000 atm).

FIG. 2A shows the chemical composition of 10,16-dihydroxyhexadecanoicacid (200 in FIG. 2A), and FIG. 2B shows the chemical composition of10,18-dihydroxyoctadecanoic acid (202 in FIG. 2B), both of which aretypical direct monomer products of the cutin thermal depolymerizationprocess carried out with water as a solvent, and form the majoritybuilding block of cutin. Other direct monomer products that can resultfrom the cutin thermal depolymerization process carried out with wateras a solvent are shown in FIGS. 2C, 2D, 2E, and 2F, where FIG. 2C showsthe chemical composition of 9,16-dihydroxyhexadecanoic acid (204 in FIG.2C), FIG. 2D shows the chemical composition of9,18-dihydroxyoctadecanoic acid (206 in FIG. 2D), FIG. 2E shows thechemical composition of 9,10,16-trihydroxyhexadecanoic acid (208 in FIG.2E), and FIG. 2F shows the chemical composition of9,10,18-trihydroxyoctadecanoic acid (210 in FIG. 2F). The exact productsthat result directly from the thermal depolymerization process depend onthe particular plant source of the cutin and the solvent in which thethermal depolymerization process is carried out. For example, cutin fromtomatoes tends to have a high proportion of C₁₆ acids (e.g., fatty acidshaving a carbon chain length of 16) such as that of FIGS. 2A, 2C, 2E,and 2G, whereas cutin from cranberries tends to have a high proportionof C₁₈ acids such as that of FIGS. 2B, 2D, and 2F. In someimplementations, the thermal depolymerization process produces compoundsof Formula I:

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², m, n, and o areas previously defined for Formula I. In some embodiments, R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² in Formula I are H.

In addition to the compounds of Formula I, as well as monomers and/oroligomers of the molecules 200, 202, 204, 206, 208, and 210 in FIGS.2A-2F, respectively, other products may also be formed during thethermal depolymerization process carried out in water that are notformed by depolymerization of cutin by other methods. For example,unsaturated products 302, 304, 306, 308, 310, 312, 314, 316, and/or 318in FIGS. 3A-3I, respectively, can be formed during the thermaldepolymerization process carried out in water. Here FIG. 3A illustrates(E)-16-hydroxyhexadec-9-enoic acid (302), FIG. 3B illustrates(E)-16-hydroxyhexadec-10-enoic acid (304), FIG. 3C illustrates(Z)-16-hydroxyhexadec-9-enoic acid (306), FIG. 3D illustrates(Z)-16-hydroxyhexadec-10-enoic acid (308), FIG. 3E illustrates10-hydroxyhexadec-15-enoic acid (310), FIG. 3F illustrates(E)-hexadeca-9,15-dienoic acid (312), FIG. 3G illustrates(E)-hexadeca-10,15-dienoic acid (314), FIG. 3H illustrates(Z)-hexadeca-9,15-dienoic acid (316), and FIG. 3I illustrates(Z)-hexadeca-10,15-dienoic acid (318). In general, the thermaldepolymerization methods described herein can produce compounds ofFormula II:

where R¹, R², R³, R^(3′), R⁴, R⁵, R⁶, R^(6′), m, and n are as previouslydefined for Formula II. In some embodiments, R¹, R², R³, R^(3′), R⁴, R⁵,R⁶, R^(6′) in Formula II are H.

In some embodiments, the thermal depolymerization methods describedherein can produce compounds of the Formula III:

wherein:

R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each independently, ateach occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl,wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, orhalogen;

R³, R⁴, R⁷, and R⁸ are each independently, at each occurrence, —H,—OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl whereineach alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, or halogen; or

R³ and R⁴ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle; and/or

R⁷ and R⁸ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle;

R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl;

the symbol

represents a single bond or a cis or trans double bond;

n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

m is 0, 1, 2 or 3;

q is 0, 1, 2, 3, 4 or 5; and

r is 0, 1, 2, 3, 4, 5, 6, 7 or 8; and

R is selected from —H, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl,—C₃-C₇ cycloalkyl, aryl, 1-glycerol, 2-glycerol, or heteroaryl.

In some embodiments, R can be —H, —CH₃, or —CH₂CH₃.

In some embodiments, the thermal depolymerization methods describedherein can produce compounds of the Formula III-A:

wherein:

each R^(a) is independently —H or —C₁-C₆ alkyl;

each R^(b) is independently selected from —H, —C₁-C₆ alkyl, or —OH;

R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each independently, ateach occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl,wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, orhalogen;

R³, R⁴, R⁷, and R⁸ are each independently, at each occurrence, —H,—OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl wherein each alkyl,alkynyl, cycloalkyl, aryl, or heteroaryl is optionally substituted withone or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, or halogen; or

R³ and R⁴ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle; and/or

R⁷ and R⁸ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle;

R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl;

the symbol

represents a single bond or a cis or trans double bond;

n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

m is 0, 1, 2 or 3;

q is 0, 1, 2, 3, 4 or 5; and

r is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the thermal depolymerization methods describedherein can produce compounds of the Formula III-B:

wherein:

each R^(a) is independently —H or —C₁-C₆ alkyl;

each R^(b) is independently selected from —H, —C₁-C₆ alkyl, or —OH;

R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each independently, ateach occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl,wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, orhalogen;

R³, R⁴, R⁷, and R⁸ are each independently, at each occurrence, —H,—OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆alkynyl, —C₃-C₇ cycloalkyl, aryl, or heteroaryl wherein each alkyl,alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with one or more —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, or halogen; or

R³ and R⁴ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle; and/or

R⁷ and R⁸ can combine with the carbon atoms to which they are attachedto form a C₃-C₆ cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-memberedring heterocycle;

R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl;

the symbol

represents a single bond or a cis or trans double bond;

n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

m is 0, 1, 2 or 3;

q is 0, 1, 2, 3, 4 or 5; and

r is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the thermal depolymerization methods describedherein can produce one or more of the following fatty acid compounds:

In some embodiments, the thermal depolymerization methods describedherein can produce one or more of the following methyl ester compounds:

In some embodiments, the thermal depolymerization methods describedherein can produce one or more of the following ethyl ester compounds:

In some embodiments, the thermal depolymerization methods describedherein can produce one or more of the following 2-glycerol estercompounds:

In some embodiments, the thermal depolymerization methods describedherein can produce one or more of the following 1-glycerol estercompounds:

In some embodiments, any of the compounds described herein (e.g.,compounds of Formula I, II, or III) can be crosslinked to create adimer, trimer, or oligomer. For instance, a dimer can have the structureset forth below:

One of skill in the art will understand that other crosslinked dimers,trimers, or oligomers can be created in accordance with the presentdisclosure. Additionally, one of skill in the art will understand thatat the temperatures and pressures used to depolymerize cutin describedherein, oxidation of primary or secondary alcohols can occur. Forexample, when carried out in the present of oxygen (e.g., air), it ispossible that an alcohol moiety can be oxidized to a correspondingketone.

Without wishing to be bound by theory, it is believed that the saturatedproducts (e.g., 200, 202, 204, 206, 208, and/or 210) are cross-linkedwithin the cutin layer and are thereby isolated into monomer and/oroligomer units directly via depolymerization reactions, whereas theunsaturated products (e.g., 302, 304, 306, 308, 310, 312, 314, 316, 318,and/or products of Formula II) are indirect byproducts that are formedby decomposition of the direct products (e.g., 200, 202, 204, 206, 208,and/or 210). The decomposition can occur, for example, if hightemperature and/or high pressure conditions are maintained for too longa period of time during the thermal depolymerization process, where thelength of time necessary to produce the unsaturated byproducts decreaseswith increasing temperature. However, it may be possible that theunsaturated products (e.g., 302, 304, 306, 308, 310, 312, 314, 316, 318,and/or products of Formula II) are present in the cutin layer andthereby become constituents of the extract composition when thecomposition is formed by the thermal depolymerization methods describedherein, but that the production of these products is suppressed orinhibited when cutin depolymerization is carried out using other methods(e.g., acidic or basic depolymerization). Or, the unsaturated productsmay be generated as part of the thermal depolymerization process.

In some implementations, it is preferable that the direct products(e.g., 200, 202, 204, 206, 208, and/or 210) of the cutindepolymerization be present in the second mixture but not the indirectunsaturated byproducts (e.g., 302, 304, 306, 308, 310, 312, 314, 316,318, and/or products of Formula II) resulting from the decomposition(e.g., elimination) of the direct products. For example, when theresulting second mixture is subsequently used to form a protectivecoating, in many cases the coating can have desirable qualities (e.g.,higher cross-link density, lower permeability to water and/or oxygen)when the second mixture includes a large fraction of saturateddepolymerization products (e.g., 200, 202, 204, 206, 208, and/or 210)while having as small a concentration as possible of the unsaturatedindirect byproducts (e.g., 302, 304, 306, 308, 310, 312, 314, 316, 318,and/or products of Formula II). As such, the conditions of the thermaldepolymerization process (e.g., solvent composition, temperature,pressure, residence time of the mixture at high temperature andpressure, and time to heat and cool the mixture) can be adjusted suchthat the mixture after depolymerization includes a large fraction of thedirect products, such as monomers 200, 202, 204, 206, 208, and/or 210(and optionally oligomers formed thereof).

In some implementations, the conversion (by mass) of cutin tocutin-derived monomer and/or oligomer units or esters (both directproducts and indirect byproducts) using depolymerization processesdescribed herein can be at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. Furthermore, of the resultingmonomer and/or oligomer and/or ester depolymerization products (bothdirect products and indirect byproducts), at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% can be directproducts such as monomers 200 and/or 202 (and/or oligomers formedthereof). The depolymerization products can be further purified, forexample by selective filtering, to form a plant extract compositionsuitable for coating applications that is a substantially purecomposition of direct depolymerization products such as monomers 200,202, 204, 206, 208, and/or 210 (and/or oligomers formed thereof), or ofesterified or glycerated compounds formed of the direct depolymerizationproducts.

The duration of time for which the mixture is held at elevatedtemperature and pressure during the thermal depolymerization process canbe at least partially determined by the specific values of the elevatedtemperature and pressure, as well as the time required to heat and coolthe mixture. Typically, lower temperatures and/or pressures requirelonger residence times to achieve a high conversion rate of cutin intomonomer and/or oligomer units. Thus it may be preferable to use highertemperatures and/or pressures in order to reduce processing times.However, too high a temperature can cause decomposition of the directmonomers/oligomers (e.g., the monomers 200, 202, 204, 206, 208, or 210of FIGS. 2A-2F, respectively) into other byproducts, as previouslydescribed (e.g., in FIG. 3), which in many cases can adversely affectthe quality of the coatings that are subsequently formed from the plantextract compositions. For example, residence times longer than one hourat 520 K can result in significant quantities of unsaturated product,and the decomposition is further accelerated at even highertemperatures.

FIGS. 4A and 4B are qualitative plots of the relative concentrations ofdirect depolymerization products (e.g., 200, 202, 204, 206, 208, and/or210) and unsaturated indirect byproducts (e.g., 302, 304, 306, 308, 310,312, 314, 316, and/or 318) in the mixture as a function of time fordifferent thermal depolymerization conditions when water is used as thesolvent. FIG. 4A corresponds to a temperature of 498 K and a pressure ofabout 22.1 atm (about 325 psi), while FIG. 4B corresponds to atemperature of 523 K and a pressure of about 37.4 atm (about 550 psi).In both cases, the cutin was obtained from tomato skins. 402 and 404represent the relative concentrations of direct depolymerization monomerproducts (e.g., monomers of 200, 202, 204, 206, 208, and/or 210) in themixtures at different times during the thermal depolymerization process,while 406 and 408 represent the relative concentrations of indirectunsaturated byproducts (e.g., 302, 304, 306, 308, 310, 312, 314, 316,and/or 318) in the mixtures at different times during the thermaldepolymerization process.

As seen in FIGS. 4A and 4B, under both conditions there is nosubstantial concentration of direct or indirect depolymerizationproducts in the mixture at time t=0 (at the onset of the thermaldepolymerization process). Referring now to FIG. 4A, at 498 K there is asteady rise in the concentration of direct depolymerization monomerproducts with time until the concentration saturates after nearly 8hours (see 402). However, if the elevated temperature is maintainedbeyond about 8 hours, the concentration of direct depolymerizationmonomer products begins to decrease, corresponding to decomposition ofthe direct depolymerization monomer products into the unsaturatedbyproducts. Accordingly, at 498 K the concentration of unsaturatedbyproducts remains low until about about 8 hours, at which point itbegins to rise as the direct depolymerization monomer products begin todecompose (see 406).

Referring now to FIG. 4B, at 523 K the concentration of directdepolymerization monomer products rises steadily and then saturatesafter about 1 hour (see 404). Soon after, the concentration of directdepolymerization monomer products begins to decrease, as the directdepolymerization monomer products begin to decompose into theunsaturated byproducts. Correspondingly, at 523 K the concentration ofunsaturated byproducts remains low until close to about 1 hour, at whichpoint it begins to rise as the direct depolymerization monomer productsbegin to decompose (see 408). For times greater than about 2 hours at523 K, the concentration of unsaturated byproducts increases to a valuegreater than that of the direct depolymerization monomer products. Assuch, at 498 K and 22.1 atm in water, substantial conversion of cutinderived from tomato skins to monomers occurs over the course of about 8hours, but at 523 K and 37.4 atm, the conversion takes less than 1 hour.

Also illustrated qualitatively in FIG. 4A is the concentration ofoligomers (n-mers) with 1<n<10 as a function of time (410). As seen,initially the oligomer concentration rises at a much faster rate thanthe monomer concentration. However, after a time much less than 8 hours,the oligomer concentration saturates and then decreases to a value muchless than the monomer concentration. Without wishing to be bound bytheory, it is believed that at 498 K and 22.1 atm in water, the cutin isinitially being broken down primarily into oligomer units (n-mers with1<n<10), and the oligomer units are subsequently broken down intomonomer units.

The rate of decomposition of the direct monomer/oligomer products (e.g.,200, 202, 204, 206, 208, or 210 in FIGS. 2A-2F, respectively) intounsaturated byproducts such as those of FIGS. 3A-3I at highertemperatures and/or pressures is further affected by the rate of heatingand/or cooling to and from the elevated temperature. In someembodiments, longer heating and/or cooling times can result in a higherrate of monomer/oligomer decomposition into unsaturated byproducts for agiven residence time at a specific elevated temperature and pressure. Assuch, in order to prevent or suppress this decomposition, it can bepreferable to heat and cool the mixture as quickly as possible. Forinstance, in some implementations heating and cooling rates greater than10° C./min, greater than 20° C./min, or greater than 40° C./min arepreferred, with faster rates being more preferable.

Other methods for subjecting the mixtures to elevated temperature and/orpressure can be used as well. In particular, methods which minimize thetemperature rise and fall times can be preferable, since they can reducethe rate of decomposition of the monomers/oligomers at highertemperatures and/or pressures and allow for better process control. Forexample, a slurry feed or continuous flow process can be used, in whichthe mixture is continuously flowed into and then out of the vesselthrough a series of valves through which the mixture can flow, while atthe same time allowing for control of the temperature and pressurewithin the vessel. Or, dielectric heating could also be used to heat themixtures.

In some implementations, it may be preferable for some percentage (e.g.,greater than 20%, greater than 40%, greater than 60%, greater than 70%,greater than 80%, or greater than 90%) of the direct depolymerizationproducts (e.g., monomers 200, 202, 204, 206, 208, and/or 210 of FIGS.2A-2F, respectively) to decompose into other monomer/oligomer byproducts(e.g., unsaturated byproducts 302, 304, 306, 308, 310, 312, 314, 316,and 318 of FIGS. 3A-3I, respectively) that do not typically result fromthe depolymerization of cutin (or cutin-containing components).Specifically, the unsaturated byproducts 302, 304, 306, 308, 310, 312,314, 316, and 318 of FIGS. 3A-3I, respectively, as well as the compoundsof Formula II, can each be isolated and further processed to form othersaturated molecules such as those shown in FIGS. 5A, 5B, and 5C, whereFIG. 5A illustrates 9,10,16-trihydroxyhexadecanoic acid (502), FIG. 5Billustrates 16-hydroxyhexadecanoic acid (504), and FIG. 5C illustratespalmitic acid (506). Similarly, unsaturated ester byproducts, such asthose shown in FIGS. 7A-7I, 8A-8I, 9A-9I, and 10A-10I and described infurther detail below, can each be isolated and further processed to formother saturated molecules such as those shown in FIGS. 6A-6H (which arealso described in further detail below). For example, referring to FIG.4B, the reaction to convert the saturated monomers to unsaturatedbyproducts can be driven to completion, for example by carrying out thethermal depolymerization process at 523 K and 37.4 atm in water fortimes greater than 2 hours. The unsaturated byproducts can then behydrogenated with a catalyst, for example Ni or Pd, to form palmiticacid 506 or 16-hydroxyhexadecanoic acid 504. Accordingly, in such aprocess, palmitic acid can be formed from non-palm sources. Theproduction of palm oil from oil palms has a large environmental impact,including deforestation and habitat loss, as well as sociologicalimpacts, since indigenous people are often displaced to make room forlarge plantations in the developing world. For these reasons, it can bedesirable to obtain palmitic acid from non-palm sources.

Alternatively, after the reaction to convert the saturated monomers tounsaturated byproducts is driven to completion, the unsaturatedbyproducts can be hydroxylated with a catalyst (e.g., osmium tetraoxideor another hydroxylation reagent) to form, for instance,9,10,16-trihydroxyhexadecanoic acid (502) or other compounds of FormulaI, where Formula I is as previously defined.

Plant extract compositions for forming protective coatings cansubsequently be formed from any of the molecules 200, 202, 204, 206,208, 210, 502, 504, or 506 in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 5A, 5B, and5C, respectively, as well as any other direct depolymerization products(e.g., compounds of Formula I). The coatings can each be formedprimarily from one of these types of molecules, or alternatively from acombination of these molecules. Alternatively, the molecules can firstbe esterified (e.g., glycerated to form 1-monoacyglycerides and/or2-monoacylglycerides), the plant extract compositions can be formed fromthe esters or glycerated molecules, and the coatings can be formed fromthe esters or glycerated molecules in the plant extract compositions.Forming coatings from plant extract compositions formed from molecules502 or from molecules 504, or from a combination of molecules 200, 202,204, 206, 208, 210, 502, 504, and 506, or from esters or glyceratedmolecules formed thereof, can allow for further control over theproperties of both the extract compositions and the coatings. Forexample, the solubility of molecules 502, 504, and 506 (and esters orglycerated molecules formed thereof) in various solvents is differentthan that for molecules 200, 202, 204, 206, 208, and 210, (and esters orglycerated molecules formed thereof). As such, a wider variety ofsolvents may be available for use in forming plant extract compositionsfrom molecules 502, 504, or 506 (or esters or glycerated moleculesformed thereof), or from combinations of molecules 200, 202, 204, 206,208, 210, 502, 504, and 506 (or esters or glycerated molecules formedthereof), than can be used for plant extract compositions which onlyinclude substantial quantities of direct byproducts of cutindepolymerization (e.g., molecules 200, 202, 204, 206, 208, and/or 210,or esters or glycerated molecules formed thereof). Furthermore,properties of the coatings formed from the plant extract can further betailored for specific applications using molecules 502, 504, and 506(and/or esters or glycerated molecules formed thereof), either alone orin combination with molecules 200, 202, 204, 206, 208, 210, and/or oneanother. For example, the crosslink density of the resulting protectivefilms can vary depending on the percent mass of each of molecules 200,202, 204, 206, 208, 210, 502, 504, and 506 (and/or esters or glyceratedmolecules formed thereof) in the plant extract compositions, that canallow for film properties such as density and permeability to bespecifically tailored for the specific application in which the film isused. Furthermore, these molecules can be chemically modified in orderto tailor their properties, e.g., solubility, stability, and filmforming properties.

As previously described, in some implementations, themolecules/compounds obtained directly from depolymerization (e.g.,compounds 200, 202, 204, 206, 208, or 210,) or indirectly throughsubsequent processing steps (e.g., compounds 502, 504, or 506) areglycerated to form 1-monoacylclyceride and/or 2-monoacylglyceridemonomers, oligomers formed thereof, or combinations. In this case, theplant extract compositions from which protective coatings aresubsequently formed include the 1-monoacylclycerides and/or2-monoacylglycerides optionally dissolved in a solvent. The differencebetween a 1-monoacylclyceride and a 2-monoacylglyceride is the point ofconnection of the glyceride group. Protective coatings formed oversubstrates such as fruits from formulations that include combinations of2-monoacylglycerides and 1-monoacylclycerides (or optionally a differentadditive in place of the 1-monoacylclycerides) have in many cases beenfound by the inventors of the disclosure to exhibit superior performancein preventing water loss and oxidation without altering the physicalappearance of the substrate.

While esters (e.g., glyceryl esters) of direct depolymerization products200 or 202 can be formed via esterification (e.g., Fischeresterification) following the thermal depolymerization processesdescribed herein, esters can alternatively be directly formed during thethermal depolymerization processes through suitable solvent selection.For example, when the thermal depolymerization of cutin is carried outin water, the resulting direct depolymerization products can be freefatty acids (e.g., compounds 200, 202, 204, 206, 208, or 210 of FIGS.2A-2F). However, thermal depolymerization of cutin in other solvents(e.g., ethanol, methanol, or glycerol) can directly result in theproduction of esters.

For example, FIGS. 6A and 6B show the chemical structure of ethyl10,16-dihydroxyhexadecanoate 600 and ethyl 10,18-dihydroxyoctadecanoate602, respectively, which are ethyl esters of respective compounds10,16-dihydroxyhexadecanoic acid (200 in FIG. 2A) and10,18-dihydroxyoctadecanoic acid (202 in FIG. 2B). Compounds 600 and/or602 can be produced directly by carrying out the thermaldepolymerization of cutin in ethanol, or alternatively by carrying outthe thermal depolymerization process in a solvent that includes ethanol,for example water with ethanol added as a co-solvent.

As another example, FIGS. 6C and 6D show the chemical structure ofmethyl 10,16-dihydroxyhexadecanoate 610 and methyl10,18-dihydroxyoctadecanoate 612, respectively, which are methyl estersof respective compounds 10,16-dihydroxyhexadecanoic acid (200 in FIG.2A) and 10,18-dihydroxyoctadecanoic acid (202 in FIG. 2B). Compounds 610and/or 612 can be produced directly by carrying out the thermaldepolymerization of cutin in methanol, or alternatively by carrying outthe thermal depolymerization process in a solvent that includesmethanol, for example in water with methanol added as a co-solvent.

As yet another example, FIGS. 6E, 6F, 6G and 6H show the chemicalstructure of 2,3-dihydroxypropyl 10,16-dihydroxyhexade canoate 620,1,3-dihydroxypropan-2-yl 10,16-dihydroxyhexadecanoate 630,2,3-dihydroxypropyl 10,18-dihydroxyoctadecanoate 622, and1,3-dihydroxypropan-2-yl 10,18-dihydroxyoctadecanoate 632, respectively.Compounds 620 and 630 are glyceryl esters of 10,16-dihydroxyhexadecanoicacid (200 in FIG. 2A), and compounds 622 and 632 are glyceryl esters of10,18-dihydroxyoctadecanoic acid (202 in FIG. 2B). Compounds 620, 630,622, and/or 632 can be produced directly by carrying out the thermaldepolymerization of cutin in glycerol, or alternatively by carrying outthe thermal depolymerization process in a solvent that includesglycerol, for example in water with glycerol added as a co-solvent.

Esters of unsaturated byproducts (e.g., compounds 302, 304, 306, 308,310, 312, 314, 316, and 318 in FIGS. 3A-3I) can also be formed viaesterification (e.g., Fischer esterification) following the thermaldepolymerization processes described herein. For example, referring toFIG. 4B, the thermal process to convert saturated monomers tounsaturated byproducts (302, 304, 306, 308, 310, 312, 314, 316, and/or318) can be driven to completion, for example by carrying out thethermal depolymerization of cutin at 523 K and 37.4 atm in water fortimes greater than 2 hours, and the unsaturated byproducts can then beesterified. Alternatively, similar to the processes described above fordirect formation of esters 600, 602, 610, 612, 620, 622, 630, and 632,esters of unsaturated byproducts can be directly formed during thethermal depolymerization processes through suitable solvent selection.Specifically, when the thermal depolymerization of cutin is carried outin water, the resulting unsaturated byproducts can be unsaturated fattyacids (e.g., compounds 302, 304, 306, 308, 310, 312, 314, 316, and/or318). However, thermal depolymerization of cutin in other solvents(e.g., ethanol, methanol, or glycerol) can directly result in theproduction of unsaturated fatty esters.

For example, FIG. 7A shows the chemical structure of ethyl(E)-16-hydroxyhexadec-9-enoate 702, FIG. 7B shows the chemical structureof ethyl (E)-16-hydroxyhexadec-10-enoate 704, FIG. 7C shows the chemicalstructure of ethyl (Z)-16-hydroxyhexadec-9-enoate 706, FIG. 7D shows thechemical structure of ethyl (Z)-16-hydroxyhexadec-10-enoate 708, FIG. 7Eshows the chemical structure of ethyl 10-hydroxyhexadec-15-enoate 710,FIG. 7F shows the chemical structure of ethyl (E)-hexadeca-9,15-dienoate712, FIG. 7G shows the chemical structure of ethyl(E)-hexadeca-10,15-dienoate 714, FIG. 7H shows the chemical structure ofethyl (Z)-hexadeca-9,15-dienoate 716, and FIG. 7I shows the chemicalstructure of ethyl (Z)-hexadeca-10,15-dienoate 718. Compound 702 is anethyl ester of compound 302, compound 704 is an ethyl ester of compound304, compound 706 is an ethyl ester of compound 306, compound 708 is anethyl ester of compound 308, compound 710 is an ethyl ester of compound310, compound 712 is an ethyl ester of compound 312, compound 714 is anethyl ester of compound 314, compound 716 is an ethyl ester of compound316, and compound 718 is an ethyl ester of compound 318. Compounds 702,704, 706, 708, 710, 712, 714, 716 and/or 718 can be produced directly bycarrying out the thermal depolymerization of cutin in ethanol at asufficiently high temperature for a sufficiently long time, oralternatively by carrying out the thermal depolymerization process in asolvent that includes ethanol, for example water with ethanol added as aco-solvent.

As another example, FIG. 8A shows the chemical structure of methyl(E)-16-hydroxyhexadec-9-enoate 802, FIG. 8B shows the chemical structureof methyl (E)-16-hydroxyhexadec-10-enoate 804, FIG. 8C shows thechemical structure of methyl (Z)-16-hydroxyhexadec-9-enoate 806, FIG. 8Dshows the chemical structure of methyl (Z)-16-hydroxyhexadec-10-enoate808, FIG. 8E shows the chemical structure of methyl10-hydroxyhexadec-15-enoate 810, FIG. 8F shows the chemical structure ofmethyl (E)-hexadeca-9,15-dienoate 812, FIG. 8G shows the chemicalstructure of methyl (E)-hexadeca-10,15-dienoate 814, FIG. 8H shows thechemical structure of methyl (Z)-hexadeca-9,15-dienoate 816, and FIG. 8Ishows the chemical structure of methyl (Z)-hexadeca-10,15-dienoate 818.Compound 802 is a methyl ester of compound 302, compound 804 is a methylester of compound 304, compound 806 is a methyl ester of compound 306,compound 808 is a methyl ester of compound 308, compound 810 is a methylester of compound 310, compound 812 is a methyl ester of compound 312,compound 814 is a methyl ester of compound 314, compound 816 is a methylester of compound 316, and compound 818 is a methyl ester of compound318. Compounds 802, 804, 806, 808, 810, 812, 814, 816 and/or 818 can beproduced directly by carrying out the thermal depolymerization of cutinin methanol at a sufficiently high temperature for a sufficiently longtime, or alternatively by carrying out the thermal depolymerizationprocess in a solvent that includes methanol, for example water withmethanol added as a co-solvent.

As yet another example, FIG. 9A shows the chemical structure of2,3-dihydroxypropyl (E)-16-hydroxyhexadec-9-enoate 902, FIG. 9B showsthe chemical structure of 2,3-dihydroxypropyl(E)-16-hydroxyhexadec-10-enoate 904, FIG. 9C shows the chemicalstructure of 2,3-dihydroxypropyl (Z)-16-hydroxyhexadec-9-enoate 906,FIG. 9D shows the chemical structure of 2,3-dihydroxypropyl(Z)-16-hydroxyhexadec-10-enoate 908, FIG. 9E shows the chemicalstructure of 2,3-dihydroxypropyl 10-hydroxyhexadec-15-enoate 910, FIG.9F shows the chemical structure of 2,3-dihydroxypropyl(E)-hexadeca-9,15-dienoate 912, FIG. 9G shows the chemical structure of2,3-dihydroxypropyl (E)-hexadeca-10,15-dienoate 914, FIG. 9H shows thechemical structure of 2,3-dihydroxypropyl (Z)-hexadeca-9,15-dienoate916, and FIG. 9I shows the chemical structure of 2,3-dihydroxypropyl(Z)-hexadeca-10,15-dienoate 918. Compound 902 is a glyceryl ester ofcompound 302, compound 904 is a glyceryl ester of compound 304, compound906 is a glyceryl ester of compound 306, compound 908 is a glycerylester of compound 308, compound 910 is a glyceryl ester of compound 310,compound 912 is a glyceryl ester of compound 312, compound 914 is aglyceryl ester of compound 314, compound 916 is a glyceryl ester ofcompound 316, and compound 918 is a glyceryl ester of compound 318.

As still another example, FIG. 10A shows the chemical structure of1,3-dihydroxypropan-2-yl (E)-16-hydroxyhexadec-9-enoate 1002, FIG. 10Bshows the chemical structure of 1,3-dihydroxypropan-2-yl(E)-16-hydroxyhexadec-10-enoate 1004, FIG. 10C shows the chemicalstructure of 1,3-dihydroxypropan-2-yl (Z)-16-hydroxyhexadec-9-enoate1006, FIG. 10D shows the chemical structure of 1,3-dihydroxypropan-2-yl(Z)-16-hydroxyhexadec-10-enoate 1008, FIG. 10E shows the chemicalstructure of 1,3-dihydroxypropan-2-yl 10-hydroxyhexadec-15-enoate 1010,FIG. 10F shows the chemical structure of 1,3-dihydroxypropan-2-yl(E)-hexadeca-9,15-dienoate 1012, FIG. 10G shows the chemical structureof 1,3-dihydroxypropan-2-yl (E)-hexadeca-10,15-dienoate 1014, FIG. 10Hshows the chemical structure of 1,3-dihydroxypropan-2-yl(Z)-hexadeca-9,15-dienoate 1016, and FIG. 10I shows the chemicalstructure of 1,3-dihydroxypropan-2-yl (Z)-hexadeca-10,15-dienoate 1018.Compound 1002 is a glyceryl ester of compound 302, compound 1004 is aglyceryl ester of compound 304, compound 1006 is a glyceryl ester ofcompound 306, compound 1008 is a glyceryl ester of compound 308,compound 1010 is a glyceryl ester of compound 310, compound 1012 is aglyceryl ester of compound 312, compound 1014 is a glyceryl ester ofcompound 314, compound 1016 is a glyceryl ester of compound 316, andcompound 1018 is a glyceryl ester of compound 318. Compounds 902, 904,906, 908, 910, 912, 914, 916, 918, 1002, 1004, 1006, 1008, 1010, 1012,1014, 1016, and/or 1018 can be produced directly by carrying out thethermal depolymerization of cutin in glycerol at a sufficiently hightemperature for a sufficiently long time, or alternatively by carryingout the thermal depolymerization process in a solvent that includesmethanol, for example water with methanol added as a co-solvent.

In some implementations, the solvent of the mixture (i.e., the solventin which the thermal depolymerization process is carried out) is aliquid at standard temperature and pressure (i.e., about 273K and about1 atmosphere), but at least partially undergoes a phase change whenbrought up to the elevated temperature and pressure. For example, incases where the solvent is water (H₂O), if the temperature of themixture is increased above 647 K and the pressure is increased above 218atm, the H₂O becomes supercritical and the thermal depolymerizationprocess is carried out in supercritical H₂O. Or, in cases where thesolvent is ethanol, if the temperature of the mixture is increased above513.9 K and the pressure is increased above 60.6 atm, the ethanolbecomes supercritical and the thermal depolymerization process iscarried out in supercritical ethanol.

Because the cutin obtained from the cutin-containing portion istypically intermixed with many of the other constituents previouslydescribed, the extract obtained from the thermal depolymerizationprocess may have a higher level of impurity constituents than can betolerated in agricultural coating applications. As such, the cutin canbe purified by selectively removing or filtering out the impurityconstituents. Selective filtering can occur either before or after thedepolymerization process, or both before and after depolymerization.Selective filtering may include one or more of the following processes:

-   -   (a) Prior to depolymerizing or partly depolymerizing the cutin,        washing and/or heating the cutin in a selective solvent for        which the solubility of impurity constituents in the selective        solvent is higher than the solubility of the cutin. In this        case, impurities are dissolved into the selective solvent,        thereby resulting in fewer impurities in the first intermediate        extract immediately after depolymerization. Examples of such a        solvent can include chloroform, diethyl ether, dichloromethane,        hexane, petroleum ether, ethyl acetate, acetone, isopropanol,        ethanol, methanol, supercritical carbon dioxide, supercritical        water, water, and mixtures thereof.    -   (b) After depolymerizing or partly depolymerizing the cutin to        obtain the second mixture comprising a first intermediate        extract including the depolymerization products in the solvent,        washing and/or heating the first intermediate extract in a        selective solvent (e.g., heptane, ethyl acetate, acetonitrile,        etc.) for which the solubility of impurity constituents in the        selective solvent is lower than the solubility of the monomers        and/or oligomers. In this case, the monomers and/or oligomers        are dissolved into the selective solvent while the impurities        are not. The impurities can then be filtered out, resulting in a        second intermediate extract dissolved in the selective solvent,        whereby the second intermediate extract has a higher purity than        the first intermediate extract. The second intermediate extract        may subsequently be solidified, e.g., by evaporating the        selective solvent.    -   (c) After depolymerizing or partly depolymerizing the cutin to        obtain the second mixture comprising a first intermediate        extract including the depolymerization products in the solvent,        washing and/or heating the first intermediate extract in a        selective solvent (e.g., chloroform or hexane) for which the        solubility of impurity constituents in the selective solvent is        higher than the solubility of the monomers and/or oligomers. In        this case, impurities are dissolved into the selective solvent,        thereby removing the impurities from the extract, and a second        intermediate extract having a higher purity than the first        intermediate extract is obtained.    -   (d) After obtaining a compound comprising cutin from a        cutin-containing portion of plant matter and prior to        depolymerizing or partly depolymerizing the cutin, exposing the        compound to supercritical carbon dioxide to selectively reduce a        concentration of at least one of, for instance, proteins,        polysaccharides, phenols, lignans, aromatic acids, terpenoids,        flavonoids, carotenoids, alkaloids, alcohols, alkanes,        aldehydes, waxes, and uncharacterized colored impurities.    -   (e) After depolymerizing or partly depolymerizing the cutin to        obtain the second mixture comprising a first intermediate        extract including the depolymerization products in the solvent,        adding a second solvent to the mixture, wherein the first and        second solvents are immiscible, the second solvent having the        property that either the impurities or the monomers/oligomers        (but not both) selectively segregate into the second solvent        over the first solvent.    -   (f) After depolymerizing or partly depolymerizing the cutin to        obtain the second mixture comprising a first intermediate        extract including the depolymerization products dissolved in the        solvent, causing the monomers/oligomers to crystallize and        filtering them from the mixture, or alternatively causing the        impurities to crystallize and filtering them from the mixture.

A protective coating can be formed from the plant extract compositionsdescribed above using the process 1100 illustrated in FIG. 11. First, asolid mixture of the monomer and/or oligomer units is dissolved in asolvent (e.g., water, ethanol, or combinations thereof) to form theplant extract composition (step 1102). The concentration of the solidmixture in the solvent can, for example, be in a range of about 0.1 to100 mg/mL. Next, the solution which includes the monomer and/or oligomerunits is applied over the surface of the substrate to be coated (step1104), for example by spray coating the substrate or by dipping thesubstrate in the solution. In the case of spray coating, the solutioncan, for example, be placed in a spray bottle which generates a finemist spray. The spray bottle head can then be held approximately six totwelve inches from the substrate, and the substrate then sprayed. In thecase of dip coating, the substrate can, for example, be placed in a bag,the solution containing the composition poured into the bag, and the bagthen sealed and lightly agitated until the entire surface of thesubstrate is wet. After applying the solution to the substrate, thesubstrate is allowed to dry until all of the solvent has evaporated,thereby allowing a coating composed of the monomer and/or oligomer unitsto form over the surface of the substrate (step 1106).

The coatings and methods described herein offer a number of distinctfeatures and advantages over current methods of maintaining freshness ofagricultural products and food. For instance, the coatings can preventwater loss and shield agricultural products from threats such asbacteria, fungi, viruses and the like. The coatings can also protect,for instance, plants and food products from physical damage (e.g.,bruising), water loss, oxidation, and photodamage. Accordingly, thecompositions, solutions, and coatings can be used to help storeagricultural products for extended periods of time without spoiling. Insome instances, the compositions and coatings allow for food to be keptfresh in the absence of refrigeration. The compositions and coatingsdescribed herein can also be edible (i.e., the coatings can be non-toxicfor human consumption). The methods for forming the coatings describedherein can be entirely organic. In some implementations, the coatingsare tasteless, colorless, and/or odorless. The coatings can be made fromthe same chemical feedstocks that are naturally found in the plantcuticle, (e.g., hydroxy and/or dihydroxy palmitic acids, and/or hydroxyoleic and stearic acids) and can thus be organic and all-natural.

In some implementations, a plant extract composition is formed fromcutin derived monomers and/or oligomers and/or esters thereof extractedfrom cutin of a first plant species (e.g., utilizing the thermaldepolymerization processes previously described), and the composition isthen disposed over plant matter of the same plant species, such that theextracted monomers and/or oligomers and/or esters form a protectivecoating over the plant matter of the first plant species. Such a coatingcan, for example, reinforce the cuticle layer that naturally exists overthe plant matter. In other implementations, a plant extract compositionis formed from cutin derived monomers and/or oligomers and/or estersextracted from cutin of a first plant species (e.g., utilizing thethermal depolymerization processes previously described), and thecomposition is then disposed over plant matter of a second plant specieswhich is different from (although in some cases could be the same as)the first plant species, such that the extracted monomers and/oroligomers and/or esters form a protective coating over the plant matterof the second plant species. For example, the plant extract compositioncan be formed from monomers and/or oligomers and/or esters extractedfrom cutin obtained from tomato or cranberry skins and then applied overstrawberries, bananas, finger limes, lemons, or other plant speciesdifferent from the plant species from which the cutin was obtained inorder to form a protective coating. As such, the protective coatingsthat are formed from the monomers and/or oligomers and/or esters of theplant extract composition can provide forms of protection against bioticand abiotic stressors for which the native cuticle layer of the secondplant species is inherently incapable of providing. For example, theprotective coatings deposited over the substrates can provide superiorprotection against water loss and oxidation than can be inherentlyprovided by the native cuticle layer. Or, the plant extract compositionscan be formulated to inhibit or provide protection against fungalgrowth, for which the native cuticle layer provides little or noprotection. In some implementations, the cutin derived monomers and/oroligomers are glycerated to form monoacylglycerides prior to thecomposition being disposed over the plant matter to form the coating.This can, for example, increase the reactivity of the monomers and/oroligomers and allow them to cross-link after being disposed over theplant matter.

In some embodiments, saturated products of the depolymerizationreactions such as, for instance, free fatty acid compounds 200 and 202in FIG. 2, can be separated or at least partially separated from theunsaturated products (or byproducts) of the depolymerization reactionsdescribed herein (e.g., unsaturated fatty acid compounds in FIGS.3A-3I). In some embodiments, the ability to separate or at leastpartially separate the different types of reaction products can be usedto purify, for instance, the saturated products (i.e., compounds whereinhydroxy groups have not been eliminated). In other words, extracting thecrude products of a hydrothermal depolymerization reaction set forthherein can be used to purify or enrich the percentage of a given productdepending on the solvent used.

As set forth in the Examples below (e.g., Examples 2, 3 and 7), in thecase of thermal depolymerization of cutin in water to generate freefatty acids, the crude isolate from a depolymerization reaction wasfirst extracted using a soxhlet apparatus and heptane. After overnightextraction and cooling of the resulting heptane, it was found that aportion of extracted product precipitated from the heptane phase. Theprecipitate was found to contain saturated products, (e.g., compounds200 and 202), unsaturated products (e.g., compounds 302 and 304), or amixture of both saturated and unsaturated products. However, it wasfound that the heptane supernatant contained depolymerization productsthat were enriched (e.g., more than 90%, more than 95%, more than 96%,more than 97%, more than 98%, or more than 99%) in unsaturated fattyacids (for example, of the type shown in FIG. 3).

Following the soxhlet extraction with heptane, the crude isolate wasagain extracted using a soxhlet apparatus and ethyl acetate. It wasfound that the resulting ethyl acetate phase contained products thatwere enriched (e.g., more than 90%, more than 95%, more than 96%, morethan 97%, more than 98%, or more than 99%) in saturated fatty acids, forinstance compounds such as 200 and 202.

FIG. 12A shows a plot of the crude isolate recovered after thermaldepolymerization in water at various temperatures and reaction timesusing 15 g tomato pomace as a starting material. The recoveries aregiven as a percentage of the original 15 g input. As shown in FIG. 12A,generally more crude isolate (i.e., cutin-derived monomers andoligomers) is recovered after longer reaction times (e.g., about 4hours) and at relatively higher temperatures (e.g., about 250° C. orgreater).

The crude isolate from FIG. 12A was extracted using a soxhlet extractorusing both heptane and ethyl acetate. FIG. 12B shows the amount ofisolate recovered after a first soxhlet extraction using heptanerelative to the initial 15 g of tomato pomace. In particular, FIG. 12Bshows the amount of isolate that precipitated from the heptane aftersoxhlet extraction. In contrast, FIG. 12C shows the amount of isolatethat remained dissolved in the heptane supernatant after soxhletextraction (relative to the 15 g of tomato pomace used as startingmaterial). Without wishing to be bound by theory, the supernatant of ahexane extraction was found to contain primarily (e.g., over 90% orsubstantially all) unsaturated free fatty acid byproducts (e.g.,compounds such as 3A-3I from FIG. 3) of the hydrothermal reactionsdescribed herein.

FIG. 12D shows the amount of isolate recovered from theheptane-extracted crude isolate after a soxhlet extraction using ethylacetate as a solvent. The amounts are given relative to the 15 g tomatopomace that was used as a starting material. Without wishing to be boundby theory, the material that was extracted using ethyl acetate was foundto contain primarily (e.g., over 90% or substantially all) saturatedfree fatty acid products (e.g., compounds such as 200 and 202 from FIG.2) of the hydrothermal reactions described herein.

FIG. 13 gives a table summarizing the products isolated from the heptaneprecipitate, heptane supernatant, and ethyl acetate supernatant aftertwo soxhlet extractions of crude isolate recovered from hydrothermaldepolymerization using heptane and ethyl acetate, respectively. Thecolumns on the left give the hydrothermal reaction conditions that wereused to depolymerize the cutin to give crude cutin isolate.

FIG. 17 shows UPLC traces of crude product recovered after hydrothermaldepolymerization at 548 K for one hour. FIG. 18 shows UPLC traces andmass spectrometry analysis of 10,16-dihydroxyhexadecanoic acid recoveredafter hydrothermal depolymerization at 548 K for one hour beforeextraction. FIG. 19 shows UPLC traces and mass spectrometry analysis ofunsaturated fatty acid recovered after hydrothermal depolymerization at548 K for one hour before extraction. FIG. 20 shows UPLC traces and massspectrometry analysis of 10,16-dihydroxyhexadecanoic acid recovered fromthe heptane precipitate after hydrothermal depolymerization at 548 K forone hour and soxhlet extraction with heptane. FIG. 21 shows UPLC tracesand mass spectrometry analysis of unsaturated fatty acid recovered fromheptane supernatant after hydrothermal depolymerization at 548 K for onehour and soxhlet extraction with heptane. FIG. 22 shows UPLC traces andmass spectrometry analysis of 10,16-dihydroxyhexadecanoic acid recoveredfrom the ethyl acetate supernatant after hydrothermal depolymerizationat 548 K for one hour and soxhlet extraction with ethyl acetate.

In addition to the products of hydrothermal depolymerization, theproducts of ethanolysis (i.e., ethyl esters) can similarly be separatedor at least partially separated using selective or at least partiallyselective extractions (e.g., soxhlet extractions) and different solvents(e.g., solvents of different polarity).

For example, as set forth in the Examples below (e.g., Examples 4, 5,and 8), the crude product isolated from ethanolysis can be soxhletextracted using heptane. In some embodiments, the ethyl esters isolatedfrom ethanolysis can be less polar than the free fatty acids isolated byhydrolysis. As set forth in the Examples below, after overnight soxhletextraction of crude ethanolysis material using heptane and subsequentcooling, it was found that a precipitate formed from the heptaneextract. The heptane precipitate was found to contain products that wereenriched (e.g., more than 90%, more than 95%, more than 96%, more than97%, more than 98%, or more than 99%) in saturated ethyl esters (e.g.,hydroxylated products such as compound 600 or compound 602). Incontrast, it was found that the supernatant contained dissolved productsthat were enriched (e.g., more than 90%, more than 95%, more than 96%,more than 97%, more than 98%, or more than 99%) in unsaturated ethylesters, for example compounds such as those set forth in FIG. 7A-FIG.7I. In some embodiments, no ethyl acetate extraction was performed onthe heptane-extracted isolate of ethanolysis. For example, in someembodiments the selective precipitation of saturated (e.g.,hydroxylated) products from the heptane extraction upon cooling wassufficient to enable purification of the hydroxylated products (e.g., byfiltration).

FIG. 14A shows a plot of the crude isolate recovered after thermaldepolymerization in ethanol at various temperatures and reaction timesusing 15 g tomato pomace as a starting material. The recoveries aregiven as a percentage of the original 15 g input. As shown in FIG. 14A,generally more crude isolate (i.e., cutin-derived monomers andoligomers) is recovered after longer reaction times (e.g., about 4hours) and at relatively higher temperatures (e.g., about 523 K orgreater).

The crude isolate from FIG. 14A was extracted using a soxhlet extractorusing heptane. FIG. 14B shows the amount of isolate recovered after asoxhlet extraction using heptane relative to the initial 15 g of tomatopomace. In particular, FIG. 14B shows the amount of isolate thatprecipitated from the heptane after soxhlet extraction and cooling. Incontrast, FIG. 14C shows the amount of isolate that remained dissolvedin the heptane supernatant after soxhlet extraction (relative to the 15g of tomato pomace used as starting material). Without wishing to bebound by theory, the supernatant of the hexane extraction was found tocontain primarily (e.g., over 90% or substantially all) unsaturatedethyl ester byproducts (e.g., compounds such as 7A-7I from FIG. 7) ofthe hydrothermal reactions described herein (i.e., the unsaturated ethylesters remained dissolved in the heptane supernatant).

Without wishing to be bound by theory, the material that precipitatedfrom the heptane after extraction (which could be isolated byfiltration) was found to contain primarily (e.g., over 90% orsubstantially all) saturated ethyl ester products (e.g., compounds suchas 600 and 602 from FIG. 6) of the hydrothermal reactions describedherein.

FIG. 15 gives a table summarizing the products isolated from the heptaneprecipitate and heptane supernatant after soxhlet extraction of crudeisolate recovered from ethanolysis depolymerization. The columns on theleft give the ethanolysis reaction conditions that were used todepolymerize the cutin to give crude cutin isolate.

FIG. 23 shows UPLC traces of crude product recovered after ethanolysisdepolymerization at 548 K for four hours. FIG. 24 shows UPLC traces andmass spectrometry analysis of ethyl 10,16-dihydroxyhexadecanoaterecovered from the heptane supernatant after ethanolysisdepolymerization at 548 K for four hours and soxhlet extraction withheptane. FIG. 25 shows UPLC traces and mass spectrometry analysis ofunsaturated fatty acid ethyl ester recovered from the heptanesupernatant after ethanolysis depolymerization at 548 K for four hoursand soxhlet extraction with heptane.

EXAMPLES

The disclosure is further illustrated by the following examples andsynthesis examples, which are not to be construed as limiting thisdisclosure in scope or spirit to the specific procedures hereindescribed. It is to be understood that the examples are provided toillustrate certain embodiments and that no limitation to the scope ofthe disclosure is intended thereby. It is to be further understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which may suggest themselves to those skilled in theart without departing from the spirit of the present disclosure and/orscope of the appended claims. All solvents and other chemical reagentswere obtained from commercial sources (e.g., Sigma-Aldrich (St. Louis,Mo.)) and were used without further purification unless noted otherwise.

The following steps have been carried out to perform one or more of thethermal depolymerization processes described herein, in which cuticularmaterial was thermally processed into free fatty acids. For each ofExamples 2-5, the complete processes were carried out multiple times inorder to collect statistically significant data. Where applicable inthese examples, the data presented is given as an average value of thevarious iterations, along with the statistical deviation from theaverage.

Example 1 Method for Preparing Tomato Pomace Prior to Depolymerization

Tomato pomace obtained from a commercial tomato processing facility wasmilled in a cutting mill, and sifted to give different particle sizedistributions (e.g. >500 μm, 250-500 μm, 125-250 μm, etc.). The fractioncorresponding to 250-500 μm sequentially underwent soxhlet extractionswith chloroform (CHCl₃) overnight and methanol overnight to remove thesurface waxes and other soluble components, followed by drying undervacuum (<1 torr). The washed pomace was then lyophilized overnight(<0.02 torr) to remove water, and then stored in a desiccator beforeuse.

Example 2 Depolymerization of Cutin in Water at 498K

15 g of the 250-500 μm extracted tomato pomace (from Example 1) wasadded to a Parr 4563B reactor with an internal volume of 600 mL. To thiswas added 375 mL of water, after which the system was sealed and thereactor mounted to the system, followed by heating to a targettemperature of about 498K. The heating time from ambient roomtemperature (typically about 298K) to the target temperature was about45 minutes. The mixture was then held at 498K for about 8 hours, afterwhich it was rapidly cooled by an internal cooling loop to a temperaturebetween 25° C. and 40° C.

After cooling, the reactor was depressurized, unmounted, and unsealed.The reactor's internal components and walls were rinsed with ethylacetate until all material was loosened from the walls, and theresulting solid/liquid mixture was filtered through a medium porosityfilter frit. The reactor body was washed with a second portion of ethylacetate, and this was filtered through the filter cake. The solid cake,giving the ‘char’ or residue material, was washed with an additionalportion of ethyl acetate. Typical total volume of the ethyl acetatewashings was about 350 mL. The water/ethyl acetate mixture collectedfrom the filtration was separated in a 1 L separatory funnel. The phaseswere separated, and the organic layer was collected and dried byaddition of magnesium sulfate before filtering through a porous frit.The ethyl acetate was then removed from the organic phase by rotaryevaporation and further dried under high vacuum (P<0.1 torr). The totalmass of the resulting crude extract composition, which included bothsaturated and unsaturated fatty acids, was on average 2.86 g [Avg.]±0.23g (n=3).

Collection of the crude extract composition was followed bypurification. First, the crude extract was dissolved in methanol, afterwhich three times the mass of Celite 545 was added. The methanol wasremoved by rotary evaporation, allowing the material to be depositedonto the Celite. This mixture was transferred to a cellulose extractionthimble, and glass wool was placed on top of the mixture. The thimblewas placed in a soxhlet extractor, flushed with nitrogen, and extractedwith 500 mL heptane overnight.

Upon cooling, some of the monomer product (e.g., 200 in FIG. 2A) orunsaturated byproduct (e.g., 302, 306, and/or 308 in FIGS. 3A-3D,respectively) precipitated out of solution. The apparatus wasdisassembled, and the extraction thimble set aside. The heptane in theround bottom, referred to as the heptane supernatant, was thenseparated, and the heptane removed by rotary evaporation. Theprecipitate and supernatant fractions were kept separate, with theprecipitate weighing 0.594 g [Avg.]±0.18 g (n=3), and the heptanesupernatant (containing only unsaturated byproducts, e.g., 302, 304)weighing 0.500 g [Avg.]±0.06 g (n=3). The extraction thimble underwentsoxhlet extraction with ethyl acetate following the same procedure asthe previously described heptane soxhlet extraction. After cooling, theapparatus was disassembled, and the ethyl acetate in the round bottomwas removed by rotary evaporation. Upon drying under high vacuum, aviscous oil was recovered having a mass of 1.20 g [Avg.]±0.21 g (n=3).Characterization showed that the product predominately comprisedcompounds 200 (FIG. 2A). Without wishing to be bound by theory, it wasfound that the different soxhlet extractions selectively isolated eitherthe monomer product or the unsaturated byproduct, based on the solventbeing used for the extraction. The unsaturated byproduct was selectivelyisolated in the heptane soxhlet's supernatant, whereas the monomerproduct was selectively isolated in the ethyl acetate soxhlet'ssupernatant.

Conversion of biomass and product characterization were performed bygravimetric, ultra-high performance liquid chromatography-electrosprayor atmospheric-pressure chemical ionization mass spectrometry, andnuclear magnetic resonance experiments. Exemplary ¹HNMR data forrecovered products across multiple depolymerizations using the aboveprocedure is given below.

10,16-dihydroxypalmtic acid: ¹H NMR (600 MHz, Methanol-d₄) δ 3.53 (td,J=6.8, 2.8 Hz, 2H), 3.49 (dq, J=7.4, 3.7 Hz, 1H), 2.26 (t, J=7.4 Hz,3H), 1.60 (q, J=7.2 Hz, 4H), 1.52 (q, J=6.9 Hz, 4H), 1.43 (tt, J=9.1,4.7 Hz, 6H), 1.39-1.22 (m, 23H).

(E/Z)-16-hydroxyhexadec-9-enoic acid: ¹H NMR (600 MHz, Methanol-d₄) δ5.44-5.29 (m, 2H), 3.53 (td, J=6.7, 2.3 Hz, 2H), 2.26 (t, J=7.5 Hz, 3H),2.03 (dd, J=8.3, 4.8 Hz, 1H), 1.97 (p, J=5.8 Hz, 3H), 1.59 (p, J=7.2 Hz,3H), 1.56-1.48 (m, 3H), 1.43 (dt, J=11.2, 4.2 Hz, 1H), 1.40-1.27 (m,20H).

Example 3 Depolymerization of Cutin in Water at 523K

15 g of the 250-500 μm extracted tomato pomace (from Example 1) wasadded to a Parr 4563B reactor with an internal volume of 600 mL. To thiswas added 375 mL of water, after which the system was sealed and thereactor mounted to the system, followed by heating to a targettemperature of about 523K. The heating time from ambient roomtemperature (typically about 298K) to the target temperature was about45 minutes. The mixture was then held at 523K for about 2 hours, afterwhich it was rapidly cooled by an internal cooling loop to a temperaturebetween 25° C. and 40° C.

After cooling, the reactor was depressurized, unmounted, and unsealed.The reactor's internal components and walls were rinsed with ethylacetate until all material was loosened from the walls, and theresulting solid/liquid mixture was filtered through a medium porosityfilter frit. The reactor body was washed with a second portion of ethylacetate, and this was filtered through the filter cake. The solid cake,giving the ‘char’ or residue material, was washed with an additionalportion of ethyl acetate. Typical total volume of the ethyl acetatewashings was about 350 mL. The water/ethyl acetate mixture collectedfrom the filtration was separated in a 1 L separatory funnel. The phaseswere separated, and the organic layer was collected and dried byaddition of magnesium sulfate before filtering through a porous frit.The ethyl acetate was then removed from the organic phase by rotaryevaporation and further dried under high vacuum (P<0.1 torr). The totalmass of the resulting crude extract composition, which included bothsaturated and unsaturated fatty acids, was 3.30 g [Avg.]±0.08 g (n=3).

Collection of the crude extract composition was followed bypurification. First, the crude extract was dissolved in methanol, afterwhich three times the mass of Celite 545 was added. The methanol wasremoved by rotary evaporation, allowing the material to be depositedonto the Celite. This mixture was transferred to a cellulose extractionthimble, and glass wool was placed on top of the mixture. The thimblewas placed in a soxhlet extractor, flushed with nitrogen, and extractedwith 500 mL heptane overnight.

Upon cooling, some of the monomer product (e.g., 200 in FIG. 2A) orunsaturated byproduct (302, 304, 306, and/or 308 in FIGS. 3A-3D,respectively) precipitated out of solution. The apparatus wasdisassembled, and the extraction thimble set aside. The heptane in theround bottom, referred to as the heptane supernatant, was thenseparated, and the heptane removed by rotary evaporation. Theprecipitate and supernatant fractions were kept separate, with theprecipitate weighing 0.800 g and the heptane supernatant (containing theunsaturated byproducts (e.g., 302, 304)) weighing 0.530 g. Theextraction thimble underwent soxhlet extraction with ethyl acetatefollowing the same procedure as previously described for the heptanesoxhlet extraction. After cooling, the apparatus was disassembled, andthe ethyl acetate in the round bottom was removed by rotary evaporation.Upon drying under high vacuum, a viscous oil was recovered having a massof 1.35 g [Avg.]±0.15 g (n=3). Without wishing to be bound by theory,characterization showed that the product predominately comprisedcompound 200 (see FIG. 2A). Conversion of biomass and productcharacterization were performed by gravimetric, ultra-high performanceliquid chromatography-electrospray or atmospheric-pressure chemicalionization mass spectrometry, and nuclear magnetic resonanceexperiments.

Example 4 Depolymerization of Cutin in Ethanol at 498K

15 g of the 250-500 μm extracted tomato pomace (from Example 1) wasadded to a Parr 4563B reactor with an internal volume of 600 mL. To thiswas added 170 mL of ethanol, after which the system was sealed and thereactor mounted to the system, followed by heating to a targettemperature of about 498K. The heating time from ambient roomtemperature (typically about 298K) to the target temperature was about45 minutes. The pressure in the sealed Parr 4563B reactor system wasabout 667 psi (about 45.4 atm). The mixture was then held at 498K forabout 16 hours, after which it was rapidly cooled by an internal coolingloop to a temperature between 25° C. and 40° C.

After cooling, the reactor was depressurized, unmounted, and unsealed.The solid/liquid mixture was filtered through a medium porosity filterfrit, and the reactor body and filter cake washed with additionalethanol until washing ran clear. Typical total volume of the washingswas about 350 mL. The ethanol was removed from the liquid portion byrotary evaporation, after which the material was subjected to highvacuum (P<0.1 torr). The total mass of the resulting crude extractcomposition, which included both saturated and unsaturated fatty acidethyl esters, was 6.79 g [Avg.]±0.14 g (n=3).

The unsaturated fatty acid ethyl esters were then separated from thecrude extract composition as follows. The resulting mixture, includingboth the saturated and unsaturated fatty acid ethyl esters, wasdissolved in ethyl acetate, and three times the mass of Celite 545powder was added. The ethyl acetate was removed by rotary evaporation,allowing the material to be deposited onto the Celite. This mixture wastransferred to a cellulose extraction thimble, and glass wool was placedon top of the mixture. The thimble was placed in a soxhlet extractor,flushed with nitrogen, and extracted with 500 mL heptane overnight.After cooling to room temperature, the system was disassembled, and theround-bottom flask was left to crystallize at room temperatureovernight. The solids were then separated by filtration through aBuchner funnel and filter paper. This solid material, which comprisedsaturated fatty acid ethyl esters but not unsaturated fatty acid ethylesters, was 2.28 g [Avg.]±0.25 g (n=3). Conversion of biomass andproduct characterization were performed by gravimetric, ultra-highliquid chromatography-electrospray or atmospheric-pressure chemicalionization mass spectrometry, and nuclear magnetic resonanceexperiments. Exemplary ¹HNMR data for recovered products across multipledepolymerizations using the above procedure is given below.

Ethyl 10,16-dihydroxyhexdecanoate: ¹H NMR (600 MHz, Chloroform-d) δ 4.11(q, J=7.1 Hz, 2H), 3.63 (t, J=6.8 Hz, 2H), 3.57 (s, 1H), 2.27 (t, J=7.6Hz, 2H), 1.66-1.51 (m, 6H), 1.49-1.25 (m, 21H), 1.24 (t, J=7.1 Hz, 3H).

Ethyl (E/Z)-16-hydroxyhexadec-9-enoate: ¹H NMR (600 MHz, Chloroform-d) δ5.45-5.27 (m, 2H), 4.11 (q, J=7.1 Hz, 2H), 3.63 (t, J=6.7 Hz, 2H), 2.27(t, J=7.6 Hz, 2H), 2.05-1.89 (m, 4H), 1.64-1.51 (m, 5H), 1.38-1.22 (m,22H).

Example 5 Depolymerization of Cutin in Ethanol at 548K

15 g of the 250-500 μm extracted tomato pomace (from Example 1) wasadded to a Parr 4563B reactor with an internal volume of 600 mL. To thiswas added 170 mL of ethanol, after which the system was sealed and thereactor mounted to the system, followed by heating to a targettemperature of about 548K. The heating time from ambient roomtemperature (typically about 298K) to the target temperature was about60 minutes. The pressure in the sealed Parr 4563B reactor system wasabout 1348 psi (about 91.7 atm). The mixture was then held at 548K forabout 2 hours, after which it was rapidly cooled by an internal coolingloop to a temperature between 25° C. and 40° C.

After cooling, the reactor was depressurized, unmounted, and unsealed.The solid/liquid mixture was filtered through a medium porosity filterfrit, and the reactor body and filter cake washed with additionalethanol until washing ran clear. Typical total volume of the washingswas about 350 mL. The ethanol was removed from the liquid portion byrotary evaporation, after which the material was subjected to highvacuum (P<0.1 torr). The total mass of the resulting crude extractcomposition, which included both saturated and unsaturated fatty acidethyl esters, was 7.95 g [Avg.]±0.25 g (n=3).

The unsaturated fatty acid ethyl esters were then separated from thecrude extract composition as follows. The resulting mixture, includingboth the saturated and unsaturated fatty acid ethyl esters, wasdissolved in ethyl acetate, and three times the mass of Celite 545powder was added. The ethyl acetate was removed by rotary evaporation,allowing the material to be deposited onto the Celite. This mixture wastransferred to a cellulose extraction thimble, and glass wool was placedon top of the mixture. The thimble was placed in a soxhlet extractor,flushed with nitrogen, and extracted with 500 mL heptane overnight.After cooling to room temperature, the system was disassembled, and theround-bottom flask was left to crystallize at room temperatureovernight. The solids were then separated by filtration through aBuchner funnel and filter paper. Without wishing to be bound by theory,this solid material, which comprised saturated fatty acid ethyl esterbut not unsaturated fatty acid ethyl esters, was 2.03 g [Avg.]±0.25 g(n=3).

The heptane supernatant was evaporated to dryness using a rotaryevaporator, before being placed under high vacuum (P<0.1 torr). Thisoily material, which was comprised of unsaturated fatty acids, was 2.89g [Avg.]±0.09 g (n=3). Conversion of biomass and productcharacterization were performed by gravimetric, ultra-high performanceliquid chromatography-electrospray or atmospheric-pressure chemicalionization mass spectrometry, and nuclear magnetic resonanceexperiments.

Example 6 Depolymerization of Cutin in Glycerol at 573K

15 g of the 250-500 μm extracted tomato pomace (from Example 1) wasadded to a Parr 4563B reactor with an internal volume of 600 mL. To thiswas added 190 mL of glycerol and 10 mL H₂O, after which the system wassealed and the reactor mounted to the system, followed by heating to atarget temperature of about 573K. The heating time from ambient roomtemperature (typically about 298K) to the target temperature was about60 minutes. The pressure in the sealed Parr 4563B reactor system wasabout 290 psi (about 19.7 atm). The mixture was then held at 573K forabout 4 hours, after which it was rapidly cooled by an internal coolingloop to a temperature between 25° C. and 40° C.

After cooling, the reactor was depressurized, unmounted, and unsealed.The components of the extract composition that remained in the liquidand solid portions of the mixture were extracted by diluting the mixturewith 300 mL of water. The desired product was then extracted with 400 mLethyl acetate. Residual glycerol was removed from the ethyl acetatemixture by washing it with 1 L of water. The ethyl acetate was removedby rotary evaporation. The final mass was recorded once the liquid wasevaporated to complete dryness. Without wishing to be bound by theory,the total mass of the resulting crude composition, which were found tobe enriched in unsaturated fatty acid glycerol esters (e.g., compounds902, 904, 906, and 908 in FIGS. 9A-9D, respectively), was 8.01 g.Conversion of biomass and product characterization was performed bygravimetric, ultra-high performance liquid chromatography-electrosprayor atmospheric-pressure chemical ionization mass spectrometry, andnuclear magnetic resonance experiments. Exemplary ¹HNMR data forrecovered products across multiple depolymerizations using the aboveprocedure is given below.

¹H NMR (600 MHz, Methanol-d₄) δ 5.41-5.31 (m, 2H), 4.14 (dd, J=11.4, 4.3Hz, 1H), 4.05 (dd, J=11.3, 6.3 Hz, 1H), 3.81 (p, J=5.6 Hz, 1H), 3.65(dtd, J=16.8, 11.6, 5.2 Hz, 2H), 2.34 (t, J=7.4 Hz, 4H), 2.03 (t, 2H),1.97 (d, J=7.2 Hz, 3H), 1.60 (s, 3H), 1.54-1.49 (m, 3H), 1.30 (m, 28H).

Example 7 Optimization of Extraction Conditions and Depolymerization ofCutin in Water

Various additional optimizations of the steps described in Examples 2-3above were also performed. For example, in addition to the temperaturesand residence times described in Examples 2-3 (thermal depolymerizationin water), cutin depolymerization in water through the methods describedherein was also carried out at 225° C. (498K) for 1 hour, 2 hours, 4hours, and 8 hours; at 250° C. (523K) for 1 hour, 2 hours, and 4 hours;and at 275° C. (548K) for 1 hour, 2 hours, and 4 hours. The percentageof saturated and unsaturated fatty acids recovered, relative to the 15 gof tomato pomace originally inserted into the reactor, was determined ateach temperature and time point and plotted in FIG. 12A. As seen in FIG.12A, the percent of crude extract recovered increases at highertemperatures and longer residence times.

Following the soxhlet purification, the quantity of direct monomerproduct (e.g., 200 in FIG. 2A) and unsaturated byproduct (e.g., 302,304, 306, and 308 in FIGS. 3A-3D, respectively) was determined. Aspreviously described and also shown below with reference to FIG. 13,either the direct monomer products, a mixture of the direct monomer andunsaturated byproducts, or the unsaturated byproducts are collected inthe heptane precipitate, whereby the relative amounts of each in theheptane precipitate depend on the temperature and residence time.Without wishing to be bound by theory, it is believed that thisvariation in precipitate composition may be due to the high temperatureof the solvent wash leading to melting of the materials and subsequentflow out of the soxhlet extractor (a non-selective process), rather thandissolution in the solvent and removal as a solution (a more selectiveprocess). This may be overcome by modified equipment design (e.g., acooled receiver), or use of pressure to modulate the boiling point ofthe solvent and consequently the temperature of the condensed solvent.FIG. 12B plots the amounts (as a percentage relative to the initial 15 gof tomato pomace) recovered from the heptane precipitate. As previouslydescribed, the unsaturated byproduct is selectively isolated in theheptane supernatant, of which the amount recovered (as a percentagerelative to the initial 15 g of tomato pomace) is shown in FIG. 12C.Recall that the ethyl acetate supernatant selectively isolates thedirect monomer product, of which the amount recovered (as a percentagerelative to the initial 15 g of tomato pomace) is shown in FIG. 12D. Ingeneral, longer residence times and higher temperatures resulted inincreased conversion to the byproducts.

As described above, the soxhlet extractions can selectively isolateeither the direct monomer product or unsaturated byproduct. Furthermore,adjusting the times and temperatures can at least partially determinewhich fatty acid product (direct monomer product or unsaturatedbyproduct) is produced. FIG. 13 is a table indicating which productswere recovered from the heptane precipitate, from the heptanesupernatant, and from the ethyl acetate supernatant at the variousdepolymerization temperatures and residence times tested. In FIG. 13,‘Sat’ refers to direct monomer products (including, for example, 200 inFIG. 2A), and ‘Unsat’ refers to unsaturated byproducts (including, forexample, any of compounds 302, 304, 306, and 308 in FIGS. 3A-3D,respectively). Referring first to the heptane precipitate products, atlower depolymerization temperatures (e.g., at 498K), only the directmonomer products were recovered for all residence times up to 8 hours(and no substantial product of any kind was recovered at a 1 hourresidence time). At intermediate depolymerization temperatures (523 K),only direct monomer products were recovered for the 1 hour residencetime, whereas a mixture of the direct monomer products and unsaturatedbyproducts were recovered for longer residence times (2 hours and 4hours). At higher depolymerization temperatures (548K), a mixture of thedirect monomer products and unsaturated byproducts were recovered forshorter residence times (1 hour and 2 hours), whereas only unsaturatedbyproducts were recovered for the longer (4 hour) residence time.

FIGS. 17-22 shows UPLC characterization data for depolymerization oftomato pomace at 548 K for 1 h in water. Referring now to the heptanesupernatant products, in which the unsaturated byproducts areselectively isolated, the unsaturated byproducts were not observed atlow depolymerization temperature (498 K) at residence times of 4 hoursor less, but were observed at 498 K for the case of an 8 hour residencetime. At intermediate and higher depolymerization temperatures (523 Kand 548 K, respectively), the unsaturated byproducts were observed forall residence times (1 hour, 2 hours, and 4 hours).

Referring now to the ethyl acetate supernatant products, in which thedirect monomer products are selectively isolated, the direct monomerproducts were not observed at low depolymerization temperature (498 K)at residence times of 2 hours or less, but were observed at 498 K forthe cases of 4 hour and 8 hour residence times. At intermediatedepolymerization temperatures (523 K), the direct monomer products wereobserved for all residence times (1 hour, 2 hours, and 4 hours). Athigher depolymerization temperatures (548 K), the direct monomerproducts were observed for 1 hour and 2 hour residence times, but werenot observed in the case of a 4 hour residence time.

Example 8 Optimization of Extraction Conditions and Depolymerization ofCutin in Ethanol

In addition to the optimization of the steps in Examples 2-3 (thermaldepolymerization in water) described in Example 7 above, additionaloptimization of the steps described in Examples 4-5 above (thermaldepolymerization in ethanol) were also performed. For example, inaddition to the temperatures and residence times described in Examples4-5, cutin depolymerization in ethanol through the methods describedherein was also carried out at 225° C. (498K) for 2 hours and 6 hours;at 250° C. (523K) for 1 hour, 2 hours, and 4 hours; at 275° C. (548K)for 1 hour and 4 hours; and at 300° C. for 1 hour, 2 hours, and 4 hours.The percentage of saturated and unsaturated fatty acids recovered,relative to the 15 g of tomato pomace originally inserted into thereactor, was determined at each temperature and time point and plottedin FIG. 14A. As seen in FIG. 14A, the percent of crude extract recoveredincreases at higher temperatures and longer residence times.

Following the heptane soxhlet purification procedure, the respectivequantity of direct monomer ethyl ester product (e.g., 600 in FIG. 6A)and unsaturated ethyl ester byproduct (e.g., 702, 704, 706, and 708 inFIGS. 7A-7D, respectively) was determined. As previously described, thedirect monomer products are collected in the heptane precipitate,thereby separating the direct monomer products from the unsaturatedbyproducts. FIG. 14B plots the amounts (as a percentage relative to theinitial 15 g of tomato pomace) recovered from the heptane precipitate.As previously described, the unsaturated byproduct is selectivelyisolated in the heptane supernatant, of which the amount recovered (as apercentage relative to the initial 15 g of tomato pomace) is shown inFIG. 14C. In general, longer residence times and higher temperaturesresulted in increased conversion to desired products.

As described above, the soxhlet extractions can selectively isolateeither the direct monomer ester product or unsaturated ester byproduct.Furthermore, adjusting the times and temperatures can at least partiallydetermine which fatty acid ester product (direct monomer ester productor unsaturated ester byproduct) is produced. FIG. 15 is a tableindicating which products were recovered from the heptane precipitateand from the heptane supernatant at the various depolymerizationtemperatures and residence times tested. In FIG. 15, ‘Sat’ refers todirect monomer ester products (e.g., 600 in FIG. 6A) and ‘Unsat’ refersto unsaturated ester byproducts (including, for example, any ofcompounds 702, 704, 706, and 708 in FIGS. 7A-7D, respectively).Referring first to the heptane precipitate products, at lowerdepolymerization temperatures (e.g., at 498 K), only the direct monomerproducts were recovered for residence times of at least 6 hours (and nosubstantial product of any kind was recovered at 2 hours residencetime). At higher depolymerization temperatures (523K, and 573K), directmonomer product was recovered for residence times of 1 hour, 2 hours,and 4 hours. At a depolymerization temperature of 548K, direct monomerproduct was recovered for residence times of 1 hour, 2 hours, 4 hours,and 8 hours.

Referring now to the heptane supernatant products, in which theunsaturated byproducts are selectively isolated, the unsaturatedbyproducts were not observed at low depolymerization temperature (498K)at residence times of 2 hours, 4 hours, or 16 hours. At intermediatedepolymerization temperatures (523K), the unsaturated byproducts werenot observed at residence times of 2 hours or less, but were observed at523K for the case of a 4 hour residence time. At higher depolymerizationtemperatures (548K and 573K) the unsaturated byproducts were observedfor all residence times (1 hour, 2 hours, and 4 hours).

FIGS. 23-25 give UPLC characterization data after depolymerization oftomato pomace at 548K for 4 hr in ethanol.

Example 9 Protective Coatings Formed Over Avocados

Protective coatings were formed over avocados by extracting compounds bymethod described herein (thermal depolymerization of cutin) anddepositing the compounds over the outer surface of the (unpeeled)avocados. The mass loss rates of the avocados were then measured andcompared to those of uncoated avocados. All coatings were formed bydipping the avocados in a solution comprising the associated compoundsdissolved in substantially pure ethanol at a concentration of 10 mg/mL,placing the avocados on drying racks, and allowing the avocados to dryunder ambient room conditions at a temperature in the range of about 23°C.-27° C. and humidity in the range of about 40%-55%. The avocados wereheld at these same temperature and humidity conditions for the entireduration of the time they were tested.

Mass loss rates are shown in FIG. 16 for untreated (e.g., uncoated)avocados in bar 1602, for avocados coated with ethyl10,16-dihydroxyhexadecanoate (referred to as “EtDHPA”) in bar 1604, andfor avocados coated with a mixture of EtDHPA and1,3-dihydroxypropan-2-yl hexadecanoate (referred to as “PA-2G”) in bar1606, where for bar 1606 the EtDHPA and PA-2G were mixed at a mass ratioof 30:70. For bars 1604 and 1606, the EtDHPA was formed by thermaldepolymerization of cutin in ethanol as described herein, while thePA-2G was formed by a multi-step process utilizing palmitic acid as thestarting material, where the process is described in detail in U.S.patent application Ser. No. 15/530,403 titled “PRECURSOR COMPOUNDS FORMOLECULAR COATINGS.” Each bar in the graph represents a group of 30avocados. All avocados were obtained from the same source, were pickedat about the same time, and were at a similar stage of ripening.

As seen in FIG. 16, the mass of the uncoated avocados (bar 1602)decreased at an average rate of about 1.21% per day, the mass of theavocados coated with EtDHPA (bar 1604) decreased at an average rate ofabout 1.06% per day, and the mass of the avocados coated with theEtDHPA/PA-2G mixture (bar 1606) decreased at an average rate of about0.94% per day. As such, the average mass loss rate of avocados coatedwith EtDHPA (bar 1604) was reduced by more than 12% as compared to theuncoated avocados (bar 1602), and the average mass loss rate of avocadoscoated with the EtDHPA/PA-2G mixture (bar 1606) was reduced by more than22% as compared to the uncoated avocados (bar 1602).

Various implementations of the compositions and methods have beendescribed above. However, it should be understood that they have beenpresented by way of example only, and not limitation. Where methods andsteps described above indicate certain events occurring in certainorder, those of ordinary skill in the art having the benefit of thisdisclosure would recognize that the ordering of certain steps may bemodified and such modification are in accordance with the variations ofthe disclosure. The implementations have been particularly shown anddescribed, but it will be understood that various changes in form anddetails may be made. Accordingly, other implementations are within thescope of the following claims.

1. A method of preparing cutin-derived monomers, oligomers, orcombinations thereof from cutin-containing plant matter, comprising:obtaining cutin from the cutin-containing plant matter; adding the cutinto a solvent to form a first mixture, the solvent having a boiling pointat a first temperature at a pressure of one atmosphere; and heating thefirst mixture to a second temperature and second pressure, the secondtemperature being higher than the first temperature and the secondpressure being higher than one atmosphere, to form a second mixturecomprising the cutin-derived monomers, oligomers, or combinationsthereof.
 2. The method of claim 1, further comprising separating thesolvent from the cutin-derived monomers, oligomers, or combinationsthereof in the second mixture to isolate the cutin-derived monomers,oligomers, or combinations thereof.
 3. The method of claim 2, furthercomprising dissolving the isolated cutin-derived monomers, oligomers, orcombinations thereof in a second solvent.
 4. The method of claim 1,wherein at least a portion of the cutin-derived monomers, oligomers, orcombinations thereof in the second mixture are unsaturated.
 5. Themethod of claim 4, further comprising hydrogenating the unsaturatedcutin-derived monomers, oligomers, or combinations thereof.
 6. Themethod of claim 1, wherein a rate of depolymerization of the cutin inthe solvent is at least 100 times greater at the second temperature andthe second pressure than at the first temperature at one atmosphere. 7.The method of claim 1, wherein the second temperature is at least 393 K.8. The method of claim 7, wherein the second pressure is greater than 5atm.
 9. The method of claim 8, wherein the first mixture is held at thesecond temperature and second pressure for at least about two hours. 10.The method of claim 7, wherein the second pressure is sufficiently highto maintain at least a portion of the solvent in a liquid phase at thesecond temperature.
 11. The method of claim 1, wherein the solvent isselected from the group consisting of water, glycerol, methanol,ethanol, liquid CO₂, and supercritical CO₂.
 12. The method of claim 1,wherein the solvent is substantially free of added acid or base.
 13. Themethod of claim 1, wherein the process of forming the second mixtureresults in the production of both saturated and unsaturated fatty acids.14. The method of claim 13, wherein a concentration of saturated fattyacids is higher than a concentration of unsaturated fatty acids in thesecond mixture.
 15. The method of claim 1, wherein the process offorming the second mixture results in the production of both saturatedand unsaturated fatty acid esters.
 16. The method of claim 15, wherein aconcentration of saturated fatty acid esters is higher than aconcentration of unsaturated fatty acid esters in the second mixture.17. The method of claim 1, wherein a depolymerization reaction at thesecond temperature results in the cutin-derived monomers, oligomers, orcombinations thereof in the second mixture being uncharged.
 18. A methodof preparing cutin-derived monomers, oligomers, or combinations thereoffrom cutin-containing plant matter, comprising: obtaining cutin from thecutin-containing plant matter; adding the cutin to a solvent to form afirst mixture, the solvent having a boiling point at a first temperatureat a pressure of one atmosphere; and heating the first mixture to asecond temperature and second pressure, the second temperature beinghigher than the first temperature and the second pressure being higherthan one atmosphere, to form a second mixture comprising a first groupof compounds of Formula I:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are eachindependently —H, —OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —C₁-C₆ alkyl, —C₁-C₆alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or 5- to 10-memberedring heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, orhalogen; R¹³ and R¹⁴ are each independently —H, —C₁-C₆ alkyl, —C₁-C₆alkenyl, or —C₁-C₆ alkynyl; R¹¹ is —H, glyceryl, —C₁-C₆ alkyl, —C₁-C₆alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or 5- to 10-memberedring heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, orhalogen; R¹² is —OH, —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl,—C₃-C₇ cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, whereineach alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —COOH, or—COOR¹¹; and m, n, and o are each independently an integer in the rangeof 0 to 30, and 0≦m+n+o≦30.
 19. The method of claim 18, wherein formingthe second mixture further comprises producing compounds of Formula II:

wherein: R¹, R², R⁴ and R⁵ are each independently —H, —OR¹¹, —NR¹¹R¹²,—SR¹¹, halogen, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, wherein eachalkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl is optionallysubstituted with —OR¹¹, —NR¹¹R¹², —SR¹¹, or halogen; R¹¹ and R¹² areeach independently —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, or —C₁-C₆ alkynyl;the symbol

represents an optionally single or cis or trans double bond; R³ is —OHand R^(3′) is selected from the group consisting of —H, —C₁-C₆ alkyl,—C₁-C₆ alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and aryl when

between R³ and R^(3′) is a single bond, and R³ and R^(3′) are absentwhen

between R³ and R^(3′) represents a double bond; R⁶ is —OH and R^(6′) isselected from the group consisting of —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl,—C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, and —C₆-C₁₀ aryl when

between R⁶ and R^(6′) is a single bond, and R⁶ and R^(6′) are absentwhen

between R⁶ and R^(6′) represents a double bond; n is an integer in therange of 0 to 11; m is an integer in the range of 0 to 25; and 0≦m+n≦25.20. The method of claim 19, wherein a concentration of the compounds ofFormula I is greater than a concentration of the compounds of Formula IIin the second mixture.
 21. The method of claim 19, wherein aconcentration of the compounds of Formula II is greater than aconcentration of the compounds of Formula I in the second mixture. 22.The method of claim 21, further comprising modifying the compounds ofFormula II to form a second group of compounds of Formula I.
 23. Themethod of claim 18, wherein forming the second mixture further comprisesproducing compounds of Formula III:

wherein: R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are eachindependently, at each occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen,—C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl,or heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with one or more —OR¹⁴,—NR¹⁴R¹⁵, —SR¹⁴, or halogen; R³, R⁴, R⁷, and R⁸ are each independently,at each occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl,or heteroaryl wherein each alkyl, alkynyl, cycloalkyl, aryl, orheteroaryl is optionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵,—SR¹⁴, or halogen; or R³ and R⁴ can combine with the carbon atoms towhich they are attached to form a C₃-C₆ cycloalkyl, a C₄-C₆cycloalkenyl, or 3- to 6-membered ring heterocycle; and/or R⁷ and R⁸ cancombine with the carbon atoms to which they are attached to form a C₃-C₆cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-membered ring heterocycle;R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl; the symbol

represents a single bond or a cis or trans double bond; n is 0, 1, 2, 3,4, 5, 6, 7 or 8; m is 0, 1, 2 or 3; q is 0, 1, 2, 3, 4 or 5; and r is 0,1, 2, 3, 4, 5, 6, 7 or 8; and R is selected from —H, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, 1-glycerol,2-glycerol, or heteroaryl.
 24. The method of claim 23, wherein aconcentration of the compounds of Formula I is greater than aconcentration of the compounds of Formula III in the second mixture. 25.The method of claim 23, wherein a concentration of the compounds ofFormula III is greater than a concentration of the compounds of FormulaI in the second mixture.
 26. The method of claim 25, further comprisingmodifying the compounds of Formula III to form a second group ofcompounds of Formula I.
 27. A method of preparing cutin-derivedmonomers, oligomers, or combinations thereof from cutin-containing plantmatter, comprising: obtaining cutin from the cutin-containing plantmatter; adding the cutin to a solvent to form a first mixture, thesolvent having a boiling point at a first temperature at a pressure ofone atmosphere; and heating the first mixture to a second temperatureand second pressure, the second temperature being higher than the firsttemperature and the second pressure being higher than one atmosphere, toform a second mixture comprising a first group of compounds of FormulaIII:

wherein: R¹, R², R⁵, R⁶, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are eachindependently, at each occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen,—C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl,or heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with one or more —OR¹⁴,—NR¹⁴R¹⁵, —SR¹⁴, or halogen; R³, R⁴, R⁷, and R⁸ are each independently,at each occurrence, —H, —OR¹⁴, —NR¹⁴R¹⁵, —SR¹⁴, halogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl,or heteroaryl wherein each alkyl, alkynyl, cycloalkyl, aryl, orheteroaryl is optionally substituted with one or more —OR¹⁴, —NR¹⁴R¹⁵,—SR¹⁴, or halogen; or R³ and R⁴ can combine with the carbon atoms towhich they are attached to form a C₃-C₆ cycloalkyl, a C₄-C₆cycloalkenyl, or 3- to 6-membered ring heterocycle; and/or R⁷ and R⁸ cancombine with the carbon atoms to which they are attached to form a C₃-C₆cycloalkyl, a C₄-C₆ cycloalkenyl, or 3- to 6-membered ring heterocycle;R¹⁴ and R¹⁵ are each independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆ alkenyl, or —C₂-C₆ alkynyl; the symbol

represents a single bond or a cis or trans double bond; n is 0, 1, 2, 3,4, 5, 6, 7 or 8; m is 0, 1, 2 or 3; q is 0, 1, 2, 3, 4 or 5; and r is 0,1, 2, 3, 4, 5, 6, 7 or 8; and R is selected from —H, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, 1-glycerol,2-glycerol, or heteroaryl.
 28. The method of claim 29, furthercomprising modifying the compounds of Formula III to form compounds ofFormula I:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are eachindependently —H, —OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —C₁-C₆ alkyl, —C₁-C₆alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or 5- to 10-memberedring heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, orhalogen; R¹³ and R¹⁴ are each independently —H, —C₁-C₆ alkyl, —C₁-C₆alkenyl, or —C₁-C₆ alkynyl; R¹¹ is —H, -glyceryl, —C₁-C₆ alkyl, —C₁-C₆alkenyl, —C₁-C₆ alkynyl, —C₃-C₇ cycloalkyl, aryl, or 5- to 10-memberedring heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl,or heteroaryl is optionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, orhalogen; R¹² is —OH, —H, —C₁-C₆ alkyl, —C₁-C₆ alkenyl, —C₁-C₆ alkynyl,—C₃-C₇ cycloalkyl, aryl, or 5- to 10-membered ring heteroaryl, whereineach alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl isoptionally substituted with —OR¹³, —NR¹³R¹⁴, —SR¹³, halogen, —COOH, or—COOR¹¹; and m, n, and o are each independently an integer in the rangeof 0 to 30, and 0≦m+n+o≦30.
 29. A method of preparing cutin-derivedmonomers, oligomers, or combinations thereof from cutin-containing plantmatter, comprising: obtaining cutin from the cutin-containing plantmatter; adding the cutin to a solvent to form a first mixture; andheating the first mixture to a temperature of at least 498K and apressure of at least 360 psi, thereby forming a second mixturecomprising the cutin-derived monomers, oligomers, or combinationsthereof.
 30. The method of claim 29, wherein the first mixture is heldat the temperature of at least 498K and the pressure of at least 360 psifor at least two hours.